Membrane scaffold proteins

ABSTRACT

The membrane scaffold proteins (MSP) of the present invention assemble with hydrophobic or partially hydrophobic proteins to form soluble nanoscale particles which preserve native structure and function; they are improved over liposomes and detergent micelles, in terms of stability and preservation of biological activity and native conformation. In the presence of phospholipid, MSPs form nanoscopic phospholipid bilayer disks, with the MSP stabilizing the particle at the perimeter of the bilayer domain. The particle bilayer structure allows manipulation of incorporated proteins in solution or on solid supports, including for use with such surface-sensitive techniques as scanning probe microscopy or surface plasmon resonance. The nanoscale particles, which are robust in terms of integrity and maintenance of biological activity of incorporated proteins, facilitate pharmaceutical and biological research, structure/function correlations, structure determinations, bioseparations, and drug discovery.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. patent applicationSer. No. 10/465,789, filed Jun. 18, 2003, now U.S. Pat. No. 7,038,958which is a Continuation-in-Part of U.S. patent application Ser. No.09/990,087, filed Nov. 20, 2001, now U.S. Pat. No. 7,048,949 whichclaims benefit of U.S. Provisional Application No. 60/252,233, filedNov. 20, 2000, and the present application claims benefit of U.S.Provisional Application 60/536,281, filed Jan. 13, 2004. All priorapplications are incorporated by reference in their entireties to theextent there is no inconsistency with the present disclosure.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

This invention was made, at least in part, with funding from theNational Institutes of Health (Grant Nos. R21GM63574, R01GM50007,R01GM31756, R01GM33775, and 5F32GM19024) and the National ScienceFoundation (Grant No. MCB 01-15068). Accordingly, the United StatesGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

The field of the present invention encompasses molecular biology andmembrane technology. Specifically, the present invention relates tomembrane scaffold proteins (MSPs), especially artificial MSPs, andmethods of using membrane scaffold proteins to stabilize, disperse andsolubilize fully or partially hydrophobic proteins including but notlimited to tethered, embedded or integral membrane proteins whilemaintaining the biological activities of those proteins or to stabilize,disperse and solubilize proteins which are purified and chemicallysolubilized, or directly from solubilized membrane fragments ormembranes into a mimic of the native membrane environment. Thehydrophobic proteins associate with the membrane scaffold proteins toform nanoscale disc-like structures termed Nanodiscs herein.

Several years ago we pursued structural and functional studies of lipidscomplexed with apolipoproteins (prepared from human plasma) andcharacterized these molecular assemblies by scanning probe microscopy,for example, using the adsorption of synthetic high density lipoproteindisks (rHDL, apo A-I) onto mica in an oriented manner (Carlson et al.,1997; Bayburt et al., 1998; Bayburt et al., 2000; Carlson et al., 2000).The diameters of the discoidal structures observed are approximately 10nm with a height of 5.5 nanometers. The 5.5 nm high topology observed ismost compatible with a single membrane bilayer epitaxially oriented onthe atomically flat mica surface (Carlson et al., 1997).

We subsequently discovered that purified membrane proteins can bereconstituted into the phospholipid bilayer domain of certain suchdiscoidal structures and studied in solution or subsequently adsorbed ona suitable surface for examination by structural or spectroscopictechniques that take advantage of a surface of oriented protein-bilayerassemblies. In the latter case, the underlying discoidal structurescontaining the membrane protein are easily recognizable and provide apoint of reference for judging the quality of the sample and images.

High-density lipoproteins (HDL) are spherical assemblies of a proteincomponent, termed apo A-l, and various phospholipids. HDL particles playan important role in mammalian cholesterol homeostasis by acting as theprimary conduit for reverse cholesterol transport (Fielding andFielding, 1991). The function of HDL as a cholesterol transporter reliesupon the activity of the HDL-associated enzyme lecithin-cholesterol acyltransferase, or LCAT (Glomset, 1968; Jonas, 1991), which mediates theinsertion of cholesterol esters into HDL lipoprotein particles. Certainportions of the apo A-I protein are required for the activity of thisenzyme (Holvoet et al., 1995). In addition, a part of the apo A-Iprotein is thought to be in a globular domain at the N-terminus, and tobe responsible for interactions with cell surface receptors. One nascentform of HDL particles has been assumed to be that of a discoid based onelectron microscopy of stained preparations (Forte et al., 1971). Ourlaboratory has confirmed this using atomic force microscopy (AFM)studies of synthetic forms of rHDL under aqueous conditions. This form,however, is not the predominant form in circulation in vivo. Rather, theapo A-I sequence appears to have evolved to stabilize the more prevalentspherical structural form.

Two general models for the nascent structure of HDL disks have beenproposed. One model has the apo A-I protein surrounding a circularbilayer section as a horizontal band or “belt” composed of a curvingsegmented alpha helical rod (Wlodawer et al., 1979). The other “picketfence” model has the protein traversing the edges of the bilayervertically in a series of helical segments (Boguski et al., 1986). Bothmodels are based primarily on indirect experimental evidence, and nothree dimensional structure of the entire particle is available todistinguish between them.

The currently accepted model is the belt model, which is consistent withsome electron microscopy and neutron scattering data (Wlodawer et al.,1979), where the helices are arranged longitudinally around the edge ofthe bilayer disks like a “belt” (Segrest et al. 1999). More recentinfrared spectroscopy studies using a new method of sample orientationfor dichroism measurements are more consistent with the belt model, incontrast to earlier studies (Wald et al., 1990; Koppaka et al., 1999).So far, there is no complete and direct evidence as to which model iscorrect, even though a low resolution x-ray crystal structure for apoA-I crystallized without lipid (Borhani et al., 1997) has been obtained.The low resolution crystal structure of an N-terminally truncated apoA-I shows a unit cell containing a tetrameric species composed of 4helical rods which bend into a horseshoe shape and which combine to givea circular aggregate about 125×80×40 Å. It was suggested that a dimericspecies in this belt conformation is capable of forming discoidalparticles.

The information collected to date concerning the reverse cholesteroltransport cycle and the maturation of HDL particles suggests that theapo A-I protein has unique properties that allow it to interactspontaneously with membranes resulting in the formation of lipoproteinparticles. Initial apo A-I lipid binding events have been proposed(Rogers et al., 1998), but the mechanism for conversion ofbilayer-associated forms to discoidal particles remains unclear. Theavailable evidence suggests that the energy of stabilization oflipid-free apo A-I is fairly low and that there is an equilibriumbetween two conformers (Atkinson and Small, 1986; Rogers et al., 1998).One conformer may be a long helical rod, and the other may be a helical“hairpin” structure about half as long. It has been suggested that thelow stabilization energy and conformational plasticity allow apo A-I tostructurally reorganize when it encounters a lipid membrane, thusfacilitating the structural changes that would have to take placein-both the membrane and the protein to produce discrete lipoproteinparticles (Rogers et al., 1998). Once these particles are formed, apoA-I may still undergo specific conformational changes that contribute tothe dynamic functionality of the lipoprotein particles and interactionwith enzymes and receptors. All of these interactions and changes takeplace at the protein-lipid interface and in specific topologiesproviding surface accessibility of critical residues. Thus, there islittle reason to believe that apo A-I itself would be ideal forgenerating a stable, nanometer size phospholipid bilayer of controlleddimension.

Different types of lipid aggregates are used for reconstitution andstudy of purified membrane proteins; these include membrane dispersions,detergent micelles and liposomes (FIG. 1). Purified systems forbiochemical and physical study require stability, which is not alwaysinherent in or is limiting in these systems. Liposomes are closedspherical bilayer shells containing an aqueous interior. Reconstitutioninto liposomes by detergent dialysis or other methods typically resultsin random orientation of the protein with respect to outer and lumenalspaces. Since ligands or protein targets are usually hydrophilic orcharged, they cannot pass through the liposomal bilayer as depicted inFIG. 1, although this may be advantageous in some instances. Since bothsides of the liposomal bilayer are not accessible to bulk solvent,coupling effects between domains on opposite sides of the bilayer aredifficult to study. Liposomes are also prone to aggregation and fusionand are usually unstable for long periods or under certain physicalmanipulations, such as stopped flow or vigorous mixing. The size ofliposomes obtained by extruding through defined cylindrical pore sizespolydisperse in size distribution rather than exhibiting a uniformdiameter.

Liposomes also may present difficulties due to light scattering, andaggregation of membrane proteins present in the bilayer andthermodynamic instability (Angrand et al., 1997; Savelli et al., 2000).The surface area of a liposome is relatively large (10⁵ to 10⁸ Å²). Toobtain liposomes with single membrane proteins incorporated requires alarge lipid to protein molar ratio.

Detergent micelles allow solubilization of membrane proteins byinteraction with the membrane-embedded portion of the protein and areeasy to use. Detergent micelles are dynamic and undergo structuralfluctuations that promote subunit dissociation and often presentdifficulty in the ability to handle proteins in dilute solutions. Anexcess of detergent micelles, however, is necessary to maintain theprotein in a non-aggregated and soluble state. Detergents can also bedenaturing and often do not maintain the properties found in aphospholipid bilayer system. Specific phospholipid species are oftennecessary to support and modulate protein structure and function(Tocanne et al., 1994). Thus, the structure, function, and stability ofdetergent solubilized membrane proteins may be called into question.Since an excess of detergent micelles is needed, protein complexes candissociate depending on protein concentration and the detergent proteinratio. By contrast, the MSP nanostructures of the present invention aremore robust structurally, having a phospholipid bilayer mimetic domainof discrete size and composition and greater stability and smallersurface area than unilamellar liposomes. The disk structures allowaccess to both sides of the bilayer like detergents, and also provide abilayer structure like that of liposomes.

There is a long felt need in the art for stable, defined compositionsfor the dispersion of membrane proteins and other hydrophobic orpartially hydrophobic proteins, such that the native activities andproperties of those proteins are preserved. Compounds other thanproteins can also be dispersed in the nanoscale particles of the presentinvention.

SUMMARY OF THE INVENTION

Membrane Scaffold Proteins (MSPs) as used herein are artificialamphiphilic proteins which self-assemble with phospholipids andphospholipid mixtures into nanometer size membrane bilayers. A subset ofthese nanometer size assemblies are discoidal in shape, and are referredto as Nanodisc structures. These nanoscale particles can be from about 5to about 500 nm, about 5 to about 100 nm, or about 5 to about 20 nm indiameter. These structures comprising phospholipid and MSP preserve theoverall bilayer structure of normal membranes but provide a system whichis both soluble in solution and which can be assembled or affixed to avariety of surfaces.

The amino acid sequences of specifically exemplified MSPs are given inSEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQID NO:29, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NOS:73-86,SEQ ID NO:91-99, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ IDNO:117, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133 ANDSEQ ID NO:135, and the corresponding sequences lacking the N-terminal 12amino acid His tag or 23 amino acid HisTEV tag portion. Also within thescope of the present invention are artificial variant MSPs havingconservative acid substitutions, insertions or deletions of up to fiveamino acids, or artificial variants having from 70 to 100% amino acidsequence identity with a specifically exemplified MSP sequence.

Within the scope of the present invention are those MSPs which contain aHis-tag and/or a His-tag TEV sequence, as well as those MSPs which arethe result of proteolytic or other cleavage to remove the “tag” portionof the protein, and which contain at the N-terminus one or more aminoacids derived from the protease recognition sequence, which may be asspecifically exemplified in the tables herein or with a functionalmodification (such as the TEV recognition sequence with either Ser orGly in the P1′ position, as discussed herein). It is also understoodthat certain amino acid substitutions at the P2′ position are permittedin the His TEV-MSPs or the proteolytic cleavage products thereof; forexample, the P2′ amino acid can be Ser, Gly, Thr, Ala, Asn, Lys or Met.MSPs with such substitutions are within the scope of the presentinvention. In certain embodiments, naturally occurring membrane scaffoldproteins, such as apolipoprotein A-1, A-II, C-I, C-II, C-III or E,apolipophorin III, among others, can be used in place of the artificialMSPs of the present invention (i.e., where the combination has not beenreported in the prior art).

Methods for recombinantly producing the artificial MSPs are also withinthe scope of the present invention. Besides the specifically exemplifiedartificial MSPs, there can be additional helical domains included withinthe primary structure of an artificial MSP, for example, those derivedfrom Apo A-II, apo C-I, apo C-II, apo C-III, apo E, apolipophorin III,myoglobin or hemoglobin. The numbers and orders of helical buildingblocks (See Table 19 for particular examples) can be varied, providedthat the self assembly function of Nanodisc formation is preserved.

The present invention further provides the use of the nanometer scalephospholipid bilayer structures or Nanodiscs formed using MSPs for theincorporation of additional hydrophobic or partially hydrophobicmolecules, including hydrophobic or partially hydrophobic proteins.Those additional proteins can be solubilized, for example, with the useof detergent, and the solubilized proteins can be added to a solution ofMSP, with or without phospholipid(s), and the nanoscale particlesself-assemble so that the MSPs and the additional “target” proteins areincorporated into a stable and soluble particle. Subsequently, anydetergent can be removed by dialysis or treatment with such agents asion exchange resins or macroporous polymeric adsorbent beads, e.g.,Biobeads made of styrene divinylbenzene.

Detergents (or other solubilizing agents) useful in the dispersion ofMSPs, membrane fragments, membranes or preparations of purified orpartially purified hydrophobic or partially hydrophobic proteinsinclude, without limitation, cholic acid, neutralized cholic acid,deoxycholic acid, sodium deoxycholate, includingn-dodecyl-β-D-maltoside, t-octylphenoxypolyethoxyethanol (Triton X-100,Union Carbide Chemicals and Plastics Co., Inc.),n-octyl-beta-D-glucopyranoside (octylglucoside), octaethylene glycolmonododecyl ether (C12E8), nonaethylene glycol monododecyl ether(C12E9), Emulgen 913, myristoyl sulfobetaine, dihexanoylphosphatidylcholine, digitonin and JB3-14. Peptidetergents can be usedas well. High hydrodynamic pressures (from about 200 to about 200,000atmospheres) can also be used to solubilize (solvate) hydrophobic orpartially hydrophobic proteins or other molecules.

Phospholipids which can be used in the Nanodisc assembly methods of thepresent invention include, without limitation, PC, phosphatidyl choline;PE, phosphatidyl ethanolamine, PI, phosphatidyl inositol; DPPC,dipalmitoyl-phosphatidylcholine; DMPC, dimyristoyl phosphatidyl choline;POPC, 1-palmitoyl-2-oleoyl-phosphatidyl choline; DHPC, dihexanoylphosphatidyl choline, dipalmitoyl phosphatidyl ethanolamine, dipalmitoylphosphatidyl inositol; dimyristoyl phosphatidyl ethanolamine;dimyristoyl phosphatidyl inositol; dihexanoyl phosphatidyl ethanolamine;dihexanoyl phosphatidyl inositol; 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoyl-phosphatidyl inositol; among others.The phospholipids can contain glycerol backbones or they can includesphingolipids. Generally, the phospholipid has two saturated fatty acidsof from 6 to 20 carbon atoms with a commonly used head group exemplifiedby, but not limited to, phosphatidyl choline, phosphatidyl ethanolamineand phosphatidyl serine. The head group can be uncharged, positivelycharged, negatively charged or zwitterionic. The phospholipids can benatural (those which occur in nature) or synthetic (those which do notoccur in nature), or mixtures of natural and synthetic. Desirably themolar ratio of MSPs to total membrane protein is that which producesabout 100 to about 200 phospholipid molecules in each discoidalstructure of about 10 nm in diameter. Those proteins, found in nature orassociated with the various membrane structures of a living organism,are solubilized in the MSP supported nanobilayer or Nanodisc through theprocess of self-assembly, and the native structure and activity of thetarget protein are preserved in these MSP-supported structures.

Besides purified or solubilized hydrophobic or partially hydrophobicproteins, hydrophobic or partially hydrophobic proteins bound to orwithin membranes or membrane fragments or disrupted membranes can beassembled with the MSPs of the present invention, without the need forpre-purification of the target protein. It is understood by the skilledartisan that the properties of a particular phospholipid determine itssuitability for a particular application of a Nanodisc; for example,DPPC, which is gel-like in consistency, is not an appropriate choice foruse in certain applications. Where membrane proteins are incorporatedinto Nanodiscs directly from intact or solubilized membranes or membranefragments, the use of MSP1 is preferred over MSP2.

The MSP supported bilayers or Nanodiscs can be used in solutions orapplied to a number of surfaces, such that the native structure andligand binding, antigenic determinants and/or enzymatic activities ofthe protein incorporated in the MSP supported structure are maintained.As specifically exemplified, the MSP supported structures are affixed toa gold surface, e.g., for use in surface plasmon resonance technologies,to a multiwell plate or to solid surfaces including but not limited to,beads, magnetic particles, chromatography matrix materials and others.Solid materials to which the MSPs can be affixed include, but are notlimited to, gold, silicon, polystyrene, quartz, silica, silicon oxides,silicon nitride, and other simple or complex materials. Where apolyhistidine sequence (His tag) is retained as part of the MSPs, theNanodiscs can be bound to a nickel-NTA-coated surface, for example.Other oligopeptide tags which mediate binding to a surface or facilitatepurification and which can be fused to a protein of interest, generallyat the N- or C-terminus, by such techniques include, without limitation,strep-tag (Sigma-Genosys, The Woodlands, Tex.) which directs binding tostreptavidin or its derivative streptactin (Sigma-Genosys); aglutathione-S-transferase gene fusion system which directs binding toglutathione coupled to a solid support (Amersham Pharmacia Biotech,Uppsala, Sweden); a calmodulin-binding peptide fusion system whichallows purification using a calmodulin resin (Stratagene, La Jolla,Calif.); and a maltose binding protein fusion system allowing binding toan amylose resin (New England Biolabs, Beverly, Mass.).

It is noted that a His or other tag does not interfere with formation ofhelical domains and the ability to mediate assembly of a Nanodiscparticle, nor is it required for helix formation and particle assembly.With appropriate modification of the MSP primary sequence, thepolyhistidine or other tag) portion can be removed by specificproteolytic cleavage, for example using the Tobacco Etch Virus protease,where there is cognate recognition sequence between the tag and thefirst helical domain of the MSP.

The present invention further relates to methods for the incorporationof membrane-associated or other hydrophobic or partially hydrophobicproteins (or other hydrophobic or partially hydrophobic molecules) intonanoscale lipid bilayers or Nanodiscs comprising at least one MSP of thepresent invention. Membrane proteins (tethered, embedded or integral)can be used in the methods of the present invention. These proteins canbe incorporated into nanoscale particles with MSPs from solubilizedintact membrane preparations, intact cells (native or recombinant) orfrom disrupted membranes or membrane fragments, without prepurificationor prefractionation of the membrane proteins, or the proteins can bepurified prior to incorporation (with solubilization if needed).

Tethered membrane proteins, which are associated with the membranebilayer via a relatively small portion of the protein, can beexemplified by cytochrome P450 reductases and cytochrome b5 proteinsfrom various sources.

Embedded membrane proteins have a more extensive association with thebilayer, but typically the bulk of the protein is in contact with theextracellular environment or the cytoplasm. Examples of embeddedmembrane proteins include, without limitation, the general class ofmembrane associated cytochromes P450, for example, cytochrome P450 2B4from rabbit liver microsomes, cytochrome P450 3A4 from human livermicrosomes and cytochrome P450 6B1 from insect microsomes.

The integral membrane proteins are exemplified by the general class ofproteins which include helical segments in the membrane bilayer, such asthe 7-helix transmembrane proteins, including, but not limited to,bacteriorhodopsin (bR) from Halobacterium halobium, the humanβ-adrenergic receptor, the 5-hydroxy tryptamine 1A G-protein coupledreceptor from Homo sapiens and other G-protein coupled protein receptorsfrom human, plant, animal or other sources. In general an integralmembrane protein has at least one portion which extends through themembrane bilayer. Other examples include, without limitation,channel-forming proteins, transporter proteins, signaling proteins,cytokine receptors (e.g., tumor necrosis factor receptors), interleukinreceptors, Fas receptor, CD27, CD40, CD30, insulin and insulin familyreceptors, dopamine receptors, the lysophosphatidic acid receptors, andthe chemokine receptors, such as CXCR4 and CCR5, dopamine receptors, andgrowth factor receptors (e.g., epidermal growth factor and/or HGFreceptors). There can be from one to more than twenty domains of theprotein passing through the membrane bilayer. An example of a one-passprotein which has been successfully incorporated into the nanoscaleparticles of the present invention is the aspartate receptor (Tar) fromEscherichia coli; and an example of a twenty six-pass proteinincorporated into Nanodiscs is an E. coli transhydrogenase. Members ofeach type of membrane protein have been successfully incorporated intothe nanoscale structures using the MSPs and methods of the presentinvention. In particular, cell surface receptors, and especiallyG-protein coupled receptors, including but not limited to,beta-adrenergic, chemokine and other receptor proteins, can beincorporated into nanobilayer bilayer structures formed with themembrane scaffold proteins (MSPs) of the present invention. Where it isdesired that a dimer or higher oligomer of a 7-helix transmembraneprotein is incorporated into a Nanodisc, a Nanodisc of greater than 9 nmin diameter is preferred, which can be accomplished by the use of arelatively longer MSP sequence such as MSP1E1, MSP1E2 or MSP1E3.

The present invention further provides materials and methods usingartificial or naturally occurring MSPs which increase the stability andmonodispersity of the self-assembled nanoparticles. G-protein coupledreceptors (GPCRs) are an important and diverse class of pharmaceuticaltargets in mammalian cellular membranes where they function as signaltransducing elements, bind several classes of bioactive ligands andtransmit information to the intracellular machinery. The artificial MSPsof the present invention stabilize and solubilize themembrane-associated form of GPCRs to allow purification and manipulationin solution or on a solid support for use in flow cytometry, highthroughput screening applications, on surfaces for surface-plasmonbiosensor and scanning-probe techniques, as well as other analyticalapplications. The methods for Nanodisc production of the presentinvention can be used to facilitate purification of naturally producedor recombinant membrane proteins in stable, biologically active andsoluble form.

Also within the scope of the present invention are methods for adsorbingor binding a molecule or ion of interest to a protein (or othermolecule) within a Nanodisc, where that protein (or other molecule)binds the compound or ion of interest with sufficient affinity so as topromote removal of the compound or ion of interest from a solutioncontaining it. This application of Nanodisc technology can be used toremove contaminating materials or it can be use in separation orpurification schemes. Similarly, Nanodiscs containing MSP andphospholipid can be used to separate hydrophobic materials from asolution by partitioning of the hydrophobic material into thephospholipid portion of the Nanodisc in a relatively nonspecificfashion. By way of nonlimiting example, lipophilic dyes have been shownto incorporate within Nanodiscs either during the self assembly processor by partitioning into the bilayer of the Nanodiscs from a solution.

The present invention further provides Nanodisc particles whereinproteins or carbohydrates of interest are attached (covalently ornoncovalently) to the MSPs on the exterior of the Nanodisc.Alternatively, a carbohydrate or protein can be covalently bound to analkane or phospholipid, which is then incorporated within the Nanodiscsuch that the carbohydrate is accessible to the outside, aqueousenvironment. Carbohydrates can also be in the form of glycoproteinswhich are incorporated within a Nanodisc. Such carbohydrate-carryingNanodiscs can be used to positively or negatively modulate cellularresponses, either in vivo or in vitro. This can be used also to directthe Nanodisc to a cell or surface displaying a lectin or the ligand of alectin, or a receptor of the ligand of the receptor, depending on thechoice of the carbohydrate or other molecule carried by the Nanodisc.Proteins which could be covalently or noncovalently linked to theNanodisc include, without limitation, antibodies or antigen-bindingfragments thereof, adhesins or other proteins or glycoproteins capableof binding to target molecules or cells of interest.

Yet another aspect of the present invention is the incorporation of ahydrophobic therapeutic or cosmetic molecule within the hydrophobic coreof the Nanodisc. This strategy can prolong the circulating lifetime ofthe compound and it can also provide the benefits of slow release of arelatively insoluble and/or toxic molecule. Such a hydrophobictherapeutic can include, without limitation, photodynamic therapeuticagents such psoralens, porphyrins and phthalacyanin-related molecules,tamoxifen, paclitaxel, anticancer agents such as adriamycin,daunorubicin or doxorubicin, cholesterol-lowering drugs, antibacterialagents such as vancomycin, fat soluble vitamins such as D or E, andantifungal agents such as the azoles (e.g., ketoconazole) or polyenes(e.g., Amphotericin B). The Nanodiscs into which these compounds havebeen incorporated are also within the scope of the present invention.

Other molecules which can be incorporated within Nanodiscs or attachedto Nanodiscs (such as by covalent attachment to the MSP) includeantibodies, monoclonal antibodies, antibody fragments capable of bindingto a cognate antigen, lectins, hormones, chemokines, lymphokines,peptides, lipids, albumin, amino sugars and lectins, nucleic acids,among others. Nanodiscs of the present invention can also be used tostabilize and deliver lipophilic agents which improve the appearance orquality of skin, including, but not limited to, vitamins A and/or E orretinol. Methods for improving the skin or for treating disease byadministering or applying an effective amount of a therapeutic orcosmetically active composition comprising Nanodisc particles into whichthe therapeutic or cosmetically active ingredient has been incorporatedare within the scope of the present invention. Such hydrophobic agentsare packaged within Nanodiscs either directly or through self assemblywith the hydrophobic (lipophilic) small molecule.

Drugs (therapeutic agents) discussed herein are exemplary, and are notmeant to be limiting in any way. Hydrophobic anti-inflammatory agentsinclude, but are not limited to, any known hydrophobic non-steroidalantiinflammatory agent, and any known hydrophobic steroidalantiinflammatory agent, any known non-steroidal antiinflammatory agentsuch as salicylic acid derivatives (aspirin), para-aminophenolderivatives (e.g., acetaminophen), indole and indene acetic acids(indomethacin), heteroaryl acetic acids (ketorolac), arylpropionic acids(ibuprofen), anthranilic acids (mefenamic acid), enolic acids (oxicams)and alkanones (nabumetone) and any known steroidal antiinflammatoryagent which can include corticosteriods and biologically activesynthetic analogs with respect to their relative glucocorticoid(metabolic) and mineralocorticoid (electrolyte-regulating) activities.Additionally, other drugs used in the therapy of inflammation oranti-inflammatory agents to be incorporated into Nanodiscs can include,but are not limited to, the autocoid antagonists such as all histamineand bradykinin receptor antagonists, leukotriene and prostaglandinreceptor antagonists, and platelet activating factor receptorantagonists.

Antimicrobial agents include, without limitation, antibacterial agents,antiviral agents, antifungal agents, and anti-protozoan agents.Non-limiting examples of antimicrobial agents (antibiotics) aresulfonamides, trimethoprim-sulfamethoxazole, quinolones, penicillins,and cephalosporins. Antifungal agents include, without limitationazoles, and especially Amphotericin B and nystatin. Therapeuticcompounds effective against protozoans can be similarly incorporatedwithin Nanodiscs. Solubilization, reduction of potential toxicity andcontrolled release are advantages.

Antineoplastic agents include, but are not limited to, those which aresuitable for treating tumors that may be present on or within an organ(such as carcinoma, sarcoma, hematopoietic cancers, e.g., myxoma,lipoma, papillary fibroelastoma, rhabdomyoma, fibroma, hemangioma,teratoma, mesothelioma of the AV node, lymphoma, and tumors thatmetastasize to the target organ, among others) including cancerchemotherapeutic agents, a variety of which are well known in the art,such as adriamycin, daunorubicin, doxorubicin, tamoxifen and paclitaxel.Antineoplastic agents can also include antibodies specific for theneoplastic cell and antibodies to which a therapeutic radionuclide orother therapeutic agent has been bound.

Angiogenic factors (e.g., to promote organ repair or for development ofa biobypass to avoid a thrombosis) include, but are not limited to,basic fibroblast growth factor, acidic fibroblast growth factor,vascular endothelial growth factor, angiogenin, transforming growthfactors, tumor necrosis factor, angiopoietin, platelet-derived growthfactor, placental growth factor, hepatocyte growth factor, andproliferin.

Thrombolytic (clot dissolving) agents include, but are not limited to,urokinase, plasminogen activator, urokinase, streptokinase, inhibitorsof α2-plasmin inhibitor, and inhibitors of plasminogen activatorinhibitor-1, angiotensin converting enzyme (ACE) inhibitors,spironolactone, tissue plasminogen activator (tPA), an inhibitor ofinterleukin 1β-converting enzyme, anti-thrombin III, and the like.

Where the target organ is the heart, exemplary drugs for deliveryinclude, but are not necessarily limited to drugs which are poorlysoluble in water, growth factors, angiogenic agents, calcium channelblockers, antihypertensive agents, inotropic agents, antiatherogenicagents, anti-coagulants, beta-blockers, anti-arrhythmia agents, cardiacglycosides, antiinflammatory agents, antibiotics, antiviral agents andthe like.

Calcium channel blockers include, but are not limited to,dihydropyridines such as nifedipine, nicardipine, nimodipine, and thelike; benzothiazepines such as dilitazem; phenylalkylamines such asverapamil; diarylaminopropylamine ethers such as bepridil; andbenzimidole-substituted tetralines such as mibefradil. Antihypertensiveagents include, but are not limited to, diuretics, including thiazidessuch as hydroclorothiazide, furosemide, spironolactone, triamterene, andamiloride; antiadrenergic agents, including clonidine, guanabenz,guanfacine, methyidopa, trimethaphan, reserpine, guanethidine,guanadrel, phentolamine, phenoxybenzamine, prazosin, terazosin,doxazosin, propanolol, methoprolol, nadolol, atenolol, timolol,betaxolol, carteolol, pindolol, acebutolol, labetalol; vasodilators,including hydralizine, minoxidil, diazoxide, nitroprusside; andangiotensin converting enzyme inhibitors, including captopril,benazepril, enalapril, enalaprilat, fosinopril, lisinopril, quinapril,ramipril; angiotensin receptor antagonists, such as losartan; andcalcium channel antagonists, including nifedine, amlodipine, felodipineXL, isadipine, nicardipine, benzothiazepines (e.g., diltiazem), andphenylalkylamines (e.g. verapamil). Anticoagulants include, but are notlimited to, heparin, warfarin, hirudin, tick anti-coagulant peptide, lowmolecular weight heparins (such as enoxaparin, dalteparin, andardeparin), ticlopidine, danaparoid, argatroban, abciximab andtirofiban.

Antiarrhythmic agents include, but are not limited to, sodium channelblockers (e.g., lidocaine, procainamide, encainide, flecanide, and thelike), beta adrenergic blockers (e.g., propranolol), prolongers of theaction potential duration (e.g., amiodarone), and calcium channelblockers (e.g., verpamil, diltiazem, nickel choride, and the like).Delivery of cardiac depressants (e.g., lidocaine), cardiac stimulants(e.g., isoproterenol, dopamine, norepinephrine, etc.) and combinationsof multiple cardiac agents (e.g., digoxin/quinidine to treat atrialfibrillation) is possible using the Nanodiscs of the present invention.

Agents to treat congestive heart failure, include, but are not limitedto, a cardiac glycoside, inotropic agents, a loop diuretic, a thiazidediuretic, a potassium ion sparing diuretic, an angiotensin convertingenzyme inhibitor, an angiotensin receptor antagonist, anitrovasodilator, a phosphodiesterase inhibitor, a direct vasodilator,an adrenergic receptor antagonist, a calcium channel blocker, and asympathomimetic agent. Agents suitable for treating cardiomyopathiesinclude, but are not limited to, dopamine, epinephrine, norepinephrine,and phenylephrine.

Also suitable are agents that prevent or reduce the incidence ofrestenosis including, but not limited to, taxol (paclataxane) andrelated compounds; and antimitotic agents. Other compounds that can beincorporated include vitamins A, D and E and cholesterol-controllingdrugs such as the statins.

Small molecule therapeutic agents can be incorporated into the nanoscalediscoid particles of the present invention. An advantageous plasmalifetime of Nanodiscs, the rendering of partially hydrophobic compoundssoluble via the amphipathic membrane scaffold protein encircledNanodisc, and the ability for potential targeting through modificationof the MSP or phospholipid components of the Nanodisc, are importantadvantages provided. Examples 15-18 provide specific exemplificationusing Amphotericin B, ketoconazole and photodynamic agents, but othertherapeutic molecules can be incorporated using the same or similarratios and protocols. Similarly, the MSP specifically exemplified isMSP1T2, but others having the properties taught herein can substitutetherefor.

Therapeutic Nanodisc compositions are desirably maintained as stablesoluble solutions; solutions of Nanodiscs can also be lyophilized andstored as dry powders. Administration to a patient in need of theparticular therapeutic compound contained in the Nanodiscs is preferablyby a parenteral route, which can include intravenous, intraarterial,intramuscular, intradermal, subcutaneous, or there can be contact with amucosal surface, for example by aerosolization (especially dry powderdrug-Nanodisc preparations) and inhalation either intranasally or viathe lower respiratory system, in a dosage sufficient for the intendedpatient response.

Nanodiscs of the present invention can also be used to stabilize anddeliver lipophilic agents which improve the appearance and/or quality ofskin, including but not limited to vitamin E or retinol. Methods forimproving the appearance of skin or for treating disease are also withinthe scope of the present invention. Such hydrophobic agents are packagedwithin Nanodiscs either directly or through self-assembly of the lipidand phospholipid component. Administration is desirably by topical of anamount of composition comprising Nanodiscs into which the cosmeticallyactive ingredient(s) has been incorporated application to an area ofskin in need of improvement, in an amount (and at a frequency) effectivefor improving the appearance of the skin in need of improvement.

The scope of the present invention includes the use of Nanodiscs whichcarry a hydrophobic or partially hydrophobic antigen, which can be aprotein, lipopolysaccharide, lipooligosaccharide or a lipoprotein. SuchNanodiscs can be used in immunogenic compositions, for example, asvaccine components. Viral proteins of interest include, withoutlimitation, gp120 of Human Immunodeficiency Virus, envelopeglycoproteins of Herpes simplex virus or measles virus, the “spike”protein of the SARS virus, hemagglutinin ligand of influenza virus orparainfluenza virus. Exemplary bacterial antigens include, but are notlimited to, cell surface proteins such as the M6 protein or M proteinsof Streptococcus pyogenes, fimbrillin of Porphryomonas gingivalis, InIBor ActA of Listeria monocytogenes, YadA of Yersinia enterocolitica, IcsAof Shigella flexneri, invasin of Yersinia pseudotuberculosis, productsof the acf gene of Vibrio cholerae, capsular material comprising thepoly-D-glutamate polypeptide of Bacillus anthracis, fibrinogen/fibrinbinding protein of Staphylococcus aureus, V and/or W antigens ofYersinia pestis (especially from a vaccine strain such as EV76) or fromYersinia enterocolytica or Yersinia pseudotuberculosis, and flagellin orporin of Campylobacter jejeuni. Similarly, O antigens of Salmonellatyphi, Salmonella choleraesuis, Salmonella enteritidis can beincorporated into nanodiscs, using the proteins and methods describedherein.

The present invention further provides immunogenic compositionscomprising Nanodiscs into which has been incorporated at least onehydrophobic or partially hydrophobic antigen, together with apharmaceutically acceptable carrier. Optionally an adjuvant and/or animmune stimulant, such as a chemokine, can be incorporated into thecomposition. The Nanodiscs allow the stabilization and solubilization ofa hydrophobic antigen, with the maintenance of the native conformationof the antigen, and with the presentation of hydrophilic regions of theantigen exposed to the aqueous environment, leading to an improvedimmune response in the human or animal to which the immunogeniccomposition has been administered.

An additional application of the present Nanodisc technology is indiagnostic and/or imaging procedures used in medical or veterinarysettings. In this application, a targeting agent is embedded within orbound to the Nanodisc such that a binding site is accessible to theaqueous environment and an imaging compound, such as a dye, aradionuclide or a fluorescent or luminescent molecule, is incorporatedwithin the Nanodisc. Imaging techniques useful with the Nanodiscscarrying the appropriate imaging agent, as well known to the art, can beused in magnetic resonance imaging, electron paramagnetic resonanceimaging, optical imaging and ultrasound imaging. The binding site of thetargeting agent is specific for a bacterial surface antigen, a tumorantigen, or other cell surface or tissue-specific marker. The discs areallowed to assembly from an aqueous mixture comprising imaging agent,MSP and target-specific protein. As for introduction of therapeutics,the MSP is desirably antigenically neutral, i.e., it should not triggeran immunological response within a human or animal to which it isadministered.

Additional applications of antigen-containing Nanodiscs include assaykits and methods for the detection of an antibody specific for theparticular antigen in a biological sample. Detection of the antibodybound to the Nanodisc-bound antigen can be by any means known to theart. Detection of the antibody in the biological sample indicates priorexposure of the human or animal to the antigen of interest, often thisapproach is used to recognize exposure to a pathogen. The biologicalsample can be blood, serum, plasma or tissue, especially from the lymphsystem.

We have developed Nanodiscs for use in structural, biochemical andpharmaceutical strategies by engineering the scaffold protein (MSP) forgreater stability, size homogeneity through various size classes anduseful functionalities in the resultant nanoscale lipoprotein particle.These particles can include tags for purification, binding to surfacesand physical manipulation of disks such as in hydrogels or on a goldbiosensor surface, and they can serve as robust entities for rapid andreproducible assays and solution-based NMR screening and in solid stateNMR structural studies. In NMR applications, the Nanodiscs provide astable monodisperse environment for proteins and other hydrophobicmolecules of interest, especially receptor proteins for which ligandbinding is studied. For example, compounds that bind to theNanodisc-supported receptor can exhibit broadened signals and hence, adifference spectrum between +/− target ligand can reveal the identity ofbound ligands. The nanoparticles and membrane protein scaffolds areuseful in biotechnology, the pharmaceutical industry as well as in basicresearch. In addition, the structural and functional principlesuncovered through our discovery and the related techniques facilitateunderstanding the interactions of proteins with lipid bilayers at themolecular level.

The Nanodiscs can contain a single type of functional protein, or, wherethe MSP and the resulting Nanodiscs are large, they can containmacromolecular assemblies, for example cellular motility motors(flagella or cilia), multicomponent bioreactors such as multienzymecomplexes, energy transduction complexes, or photosynthetic complexes.Where the incorporation of macromolecular assemblies, including acombination of a cytochrome and a reductase protein, a relatively largeMSP is used to prepare the Nanodiscs, using for example, MSP1E1, MSP1E2or MSP1E3. Where an MSP larger than MSP1 is used, the results areimproved by the incorporation of a higher molar ratio of lipid to MSP(from 70:1 to 140:1, or from 90:1 to 115:1).

Also within the scope of the present invention are complexes in whichmore than one Nanodisc particles are associated with one another. Thecomplexes can have longer time in circulation in a human or animal thansingle Nanodisc particles. These associations of particles can beheldtogether by electrostatic interactions (where different portions of thehydrophilic face of the helices have different charge) or they can becovalently bonded by disulfide bonds, for example, where the hydrophilicfaces of at least some of the helices of the MSPs contain cysteineresidues, or they can be constructed through genetic engineering of MSPfusion constructs. The particles in the complex can include therapeuticcompounds, antibodies or imaging compounds. For example, gadolinium canbe incorporated into Nanodiscs for use in ischemia imaging in humans.

The present invention further encompasses compositions and methodsuseful in detoxification and/or remediation of certain chemicals, wherethe appropriate binding protein or enzyme is incorporated within aNanodisc in its bioactive conformation. An example is a cytochrome P450coding sequence which encodes a protein capable of oxidizing (and/ordehalogenating) at least one halogenated hydrocarbon or lighthydrocarbon. Examples include, but are not limited to,trichloroethylene, ethylene dibromide, chloroform, carbon tetrachloride,styrene, benzene, 1,2-dichloropropane, vinyl chloride, dichloromethane,methyl chloride, methyl chloroform, 1,2-dichloroethane,1,2-dichloropropane, perchloroethylene, dichloroethylene, vinyl bromide,acrylonitrile, vinyl carbonate, ethyl carbamate, acetaminophen andmethyl tertyl-butyl ether. The incorporation of human cytochrome P450s,either with our without membrane bound redox transfer partners, intoNanodiscs, in which the overall stoichiometry and homo- andhetero-oligomerization state can be controlled, is a significantimprovement on the crude membrane preparations now used by thepharmaceutical industry for quantitation of drug metabolism,pharmacokinetics, and metabolite toxicity studies of lead compounds anddrug candidates. In such examples, the Nanodiscs can be used in solutionor they can be covalently or noncovalently bound to a solid support.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates different types of lipid aggregatesincorporating a membrane protein. Small circles and triangles representligand for intracellular and extracellular domains of the receptorproteins, respectively.

FIG. 2 shows the wheel structure of an alpha helix, with the placementof hydrophobic and hydrophilic amino acid side chains that give thehelix its amphipathic character.

FIG. 3 is a schematic of a “belt” model of an MSP supported bilayer. Therectangles represent single helices with a diameter of about 1.5 nm anda helix length of about 3.9 nm.

FIGS. 4A-4G illustrate various engineered MSP structures, shown withpicket fence topology and helical assignments based on sequenceanalysis. FIG. 4A: MSP1 showing positions of half-repeats. Half-repeat 1is disordered based on molecular dynamics simulation (Phillips, 1997).FIG. 4B: Hinge domain movement. FIG. 4C: Removal of half-repeats. FIG.4D: Hinge domain replacement with helices 3 and 4. FIG. 4E: MSP2, with atandem duplication of the sequence of MSP1. FIG. 4F: Removal ofhalf-repeat 1 to make MSP1D1. FIG. 4G: Tandem repeat of MSP1D1 to formMSP2D1.

FIGS. 5A-5B diagrammatically illustrate the PCR strategy used to amplifyartificial MSPs.

FIGS. 6A-6B show diagrams of the tandem repeat MSP2 with a long linker(FIG. 6A) and with a short linker sequence (FIG. 6B).

FIGS. 7A-7B show the membrane proteins incorporated into disks andattached to solid supports. FIG. 7A: Disk-associated receptor andligand-induced assembly of receptor-target complex on gold. FIG. 7B:Disk-associated receptor in a gel matrix.

FIG. 8 is a chromatogram of cytochrome P450 3A4 incorporated into 10 nmbilayer disks composed of 100% DPPC as phospholipid.

FIG. 9 illustrates the results of PAGE with sample 1 (Nanodiscs preparedwith microsomal membranes from cells coexpressing cytochrome P450 6B1and NADPH P450 reductase). Sample 2 was prepared from microsome lackingexpression of CYP6B1.

FIG. 10 provides a characteristic optical spectrum of active cytochromeP450 6B1 incorporated within Nanodiscs; the characteristic peak is at450 nm. Such spectra indicate a correct thiolate heme ligation and noevidence for the presence of an inactive “P420” form of the cytochromein the solubilized membrane bilayer system.

FIG. 11 depicts a chromatogram of sample separated by a Superdex sizingcolumn. Retention times indicated rHDL particles 10 nm in size.

FIG. 12 illustrates co-incorporation of cytochrome P450 reductase andcytochrome P450 6B1 in MSP Nanodiscs. The ratio of absorbances at 456 nm(predominantly reductase) to that at 420 nm (predominantly P450) isplotted as a function of retention time. The peak at about 26 minindicates a Nanodisc population containing both reductase andcytochrome.

FIG. 13 illustrates the binding of DPPC Nanodiscs containing carboxylterminated thiols to a gold surface, as monitored by surface plasmonresonance.

FIG. 14 provides a schematic describing the formation of nanoscalesupported lipid bilayers (Nanodiscs) through self-assembly. A cellmembrane preparation containing the target membrane protein issolubilized with detergent in the presence of membrane scaffold protein(MSP) (see herein below). Upon removal of the detergent, by dialysis orBiobeads™, a soluble MSP-supported Nanodisc, is formed with the targetincorporated into the resulting phospholipid bilayer.

FIG. 15 shows the results of one dimensional SDS-PAGE of Nanodiscmixture. Lanes 1, low molecular weight markers. Lane 2 (left panel), Sf9insect cell membranes from insect cells genetically modified for theoverexpression of CYP6B1. The band at 55 kDa represents theoverexpressed target membrane-bound protein. Lane 2 (right panel)illustrates the Nanodisc mixture assembled from Sf9 insect cellmembranes overexpressing CYP6B1. MSP1 and CYP6B1 run at molecularweights of 25 kDa and 55 kDa, respectively.

FIGS. 16A-16B show the results of size exclusion chromatography ofNanodiscs made using MSP1 and containing a heterologously expressedcytochrome P450, CYP6B1. The target protein is incorporated into theNanodisc through the simple self-assembly process described in the text.FIG. 16A: Chromatogram showing the size separation of the reconstitutedparticles (Superdex™ 200). Dotted line shows size separation of amembrane sample in the absence of MSP showing the presence of highmolecular weight non-specific and non-functional aggregates. FIG. 16B:Re-chromatogram of the CYP6B1 containing fraction demonstrating thehomogeneity of the self-assembled CYP6B1-bilayer structure.

FIG. 17 shows the preservation of phospholipid content of startingmembrane preparation in the resulting soluble Nanodisc bilayers.Vertical bars represent phospholipid type determined from threereplicate samples of starting membranes or self-assembled Nanodiscs. PC:phosphatidylcholine, PE: phosphatidylethanolamine, PI:phosphatidylinositol.

FIG. 18 shows ligand binding to CYP6B1 incorporated into Nanodiscmembrane bilayers with MSP1. The characteristic “Type I” binding spectra(decrease in substrate low spin cytochrome with absorbance at about 417nm and concomitant increase in the high spin fraction absorbing at about390 nm) is obtained in microtiter plates using high-throughput platereader following incremental addition of the environmentalfuranocoumarin xanthotoxin. A dissociation constant of roughly 30 μM wascalculated.

FIG. 19 shows the stoichiometry of phospholipid:MSP for various MSPs.The H1 helix domain does not play a significant role in the formation ofthe protein “belt” surrounding the Nanodiscs. We have found that thesizes of Nanodiscs constructed with MSP1 and those Nanodiscs constructedwith MSPs missing either half or all of H1 are the same and have thesame number of phospholipid molecules incorporated per Nanodisc.

FIG. 20 shows size exclusion chromatography elution profiles for theNanodiscs self-assembled with: A—MSP1—DPPC; B—MSP1E1—DPPC;C—MSP1E2—DPPC; D—MSP1E3—DPPC. Curve E shows the elution profile of theset of calibration proteins: 1—Bovine serum albumin, 2—Bovine livercatalase, Stokes diameter 10.4 nm; 3—Ferritin, diameter 12.2 nm;4—Thyroglobulin.

FIG. 21A shows the inhibition of Candida albicans by Nanodiscs loadedwith ketoconazole and by a solution of ketoconazole in 1% DMS. There wasno inhibition by “empty” Nanodiscs. FIG. 21B shows that there is nogrowth inhibition by the Nanodisc buffer, 1% DMSO or by the lower amountof ketoconazole.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations used in this application include A, Ala, Alanine; M, Met,Methionine; C, Cys, Cysteine; N, Asn, Asparagine; D, Asp, Aspartic Acid;P, Pro, Proline; E, Glu, Glutamic Acid; Q, Gln, Glutamine; F, Phe,Phenylalanine; R, Arg, Arginine; G, Gly, Glycine; S, Ser, Serine; H,His, Histidine; T, Thr, Threonine; I, lie, Isoleucine; V, Val, Valine;K, Lys, Lysine; W, Try, Tryptophan; L, Leu, Leucine; Y, Tyr, Tyrosine;MSP, membrane scaffold protein; DPPC, dipalmitoyl phosphatidylcholine;PC, phosphatidylcholine; PS, phosphatidyl serine; BR, bacteriorhodopsin;apo A-I, apolipoprotein A-I; GABA, gamma aminobutyric acid; PACAP,pituitary adenylate cyclase-activating polypeptide.

The simplest single-celled organisms are composed of central regionsfilled with an aqueous material and a variety of soluble small moleculesand macromolecules. Enclosing this region is a membrane which iscomposed of phospholipids arranged in a bilayer structure. In morecomplex living cells, there are internal compartments and structuresthat are also enclosed by membranes. There are numerous proteinmolecules embedded or associated within these membrane structures, andthese so-called membrane proteins are often the most important fordetermining cell functions including communication and processing ofinformation and energy. The largest problem in studying membraneproteins is that the inside of the phospholipid bilayer is hydrophobicand the embedded or anchored part of the membrane protein is itself alsohydrophobic. In isolating these membrane proteins from their nativemembrane environments, it is very difficult to prevent them from formingaggregates, which may be inactive or insoluble in the aqueousenvironments commonly used for biochemical investigations. The presentinvention provides ways to generate a soluble nanoparticle that providesa native-like phospholipid bilayer into which hydrophobic proteins ofinterest (target proteins) can be incorporated to maintain the targetprotein or smaller hydrophobic molecule as a soluble and monodisperseentity. This is accomplished by incorporating hydrophobic proteins suchas membrane proteins into nanometer scale structures using the MSPs asdescribed herein.

Solubilizing Agents

In the context of the present application, a solubilizing agent is onewhich disrupts hydrophobic interactions which lead to assembly oraggregation of hydrophobic and/or amphiphilic molecules into threedimension structures. For example, a solubilizing agent such as adetergent is used to put into solution hydrophobic proteins withinmembranes or membrane fragments. Detergents useful in the presentcontext include, but are not limited to, cholate, deoxycholate,1-palmitoyl-2-oleoyl-sn-glycerophosphocholate,1-palmitoyl-2-oleoyl-sn-glycerophosphoserine,1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine, CHAPS,n-dodecyl-β-D-maltoside, octyl-glucopyranoside, Triton X-100, myristoylsulfobetaine, dihexanoyl-phosphotidylcholine, digitonin, emulgen 913 o4rJB3-14. Peptidetergents can also be used; see, for example, Schafmeisteret al. (1993).

Membrane Scaffold Proteins

Membrane Scaffold Proteins (MSPs) as used herein may be artificial(non-naturally occurring, those which do not occur in nature, i.e.,those which differ in amino acid sequence from any naturally occurringproteins) amphiphilic proteins which self-assemble with phospholipidsand phospholipid mixtures into nanometer size membrane bilayers. Asubset of these nanometer size assemblies are discoidal in shape, andare referred to as Nanodiscs or Nanodisc structures. Desirably the MSPscomprise several helical domains, where the pairs of helical domains areseparated by a punctuation region, made up of one to five amino acidswhich do not favor helix formation or which tend to stop helix formationof adjacent amino acids. Exemplary helical regions are provided in Table19. These building blocks can be combined in orders and numbers otherthan those specifically exemplified, provided that the function of selfassembly into stable, soluble nanoscale disc-like particles ismaintained. Similarly, these specifically exemplified building blockscan be combined with other helical building blocks from other proteinssuch as other apolipoproteins, apolipophorins and the like. Theseassembled structures of MSP and phospholipid preserve the overallbilayer structure of normal membranes but provide a system which is bothsoluble and can be assembled or affixed to a variety of surfaces. Anaturally occurring example of an MSP is human apo-A1. In addition, MSPscan be designed using helical segments of proteins other than humanapoprotein A-1, for example, apo A-1 of other species, or apo C, apo E,myoglobin or hemoglobin proteins of various species. Helical segmentsfrom more than one protein can be combined, with the appropriatepunctuation sequences, to form a MSP having the useful propertiesdescribed herein. Additionally, functional MSPs can be generated by denovo protein design wherein the desired traits of amphipathic helicalprotein structures are generated. It is also understood thatconservative amino acid substitutions can be made in the sequencesspecifically exemplified, with the proviso that the self-associationfunction is maintained. Such substitution variants can be termedhomologs of the specifically exemplified sequences. Various proteins ofinterest are described in Bolanos-Garcia et al. (2003) Progress inBiophys. Molec. Biol. 83:47-68.

Hydrophobic or partially hydrophobic proteins, e.g., membrane proteins,or membrane fragments can associate with these particles such that thehydrophobic proteins or membrane fragments are effectively solubilizedin a stable structure which maintains the functionality of the proteinwith respect to enzymatic activity or ligand binding. Similarly, otherhydrophobic or partially hydrophobic molecules of interest can also beincorporated within the nanoscale discoid particles of the presentinvention.

The Nanodisc particles are stable in solution or they can be fixed to asurface, advantageously in a uniform orientation with respect to thesurface. As used herein, a nanoparticle comprising MSPs (with or withoutanother hydrophobic or a partially hydrophobic protein) can be fromabout 5 to about 500 nm, desirably about 5 to about 100 nm, or about 5to about 20 nm in diameter. Nanoparticles (disks) of about 5 to about 15nm in diameter are especially useful.

It is also readily within the grasp of the skilled artisan to designMSPs for packaging hydrophobic passenger compounds, proteins orcomplexes where the MSP assumes an amphiphilic conformation based onbeta sheets, where the amino acid sequence of the protein is punctuatedso that there are regions of beta sheet forming portions separated by aflexible (hinge) region of amino acids. The region of beta sheet-formingsequence is desirably from about 10 to about 30 amino acids, and thepunctuation region can include from 3 to 10 amino acids, where there areantiparallel beta sheets in the MSP or from about 10 to about 30 aminoacids where the beta sheets are parallel.

Functional MSPs may or may not have punctuation between domains ofsecondary structure. The punctuation region disrupts regions ofsecondary structure within a protein. Proline and/or glycine residuesare preferred punctuation regions in a protein having helical domains.Besides disrupting a domain with a particular characteristic secondarystructure, the punctuation regions can provide flexibility to aprotein's structure, serving to create a hinge region, especially in thecase of two to three amino acids, desirably including proline, glycineand alanine residues. A punctuation region (or punctuation sequence,hinge region or hinge sequence) can include from 1 to 30 amino acids,desirably 1 to 2 amino acids when the domains of secondary structure arealpha helices, and, where there are antiparallel beta sheets in the MSP,5 to 30, and especially 3 to 10 amino acids.

The necessary properties of the linker (punctuation, hinge) sequencebetween fused MSPs are flexibility and solubility so that the fusedproteins assemble into particles in a manner similar to two separate MSPmolecules. Linker sequences consisting of repeats ofGly-Gly-Gly-Ser/Thr-(SEQ ID NO:46) have these properties. It is alsodesirable, in at least some MSPs, to minimize the length of the linker.We constructed a fusion with the minimal linker sequence-GT-, whichcorresponds to the consensus DNA restriction site for Kpn I, asdescribed herein below. The Kpn I site provides an easy way of insertingany desired linker sequence by restriction with Kpn I and insertion ofdouble-stranded synthetic DNA encoding any desired linker (Robinson etal. 1998). We have also made a fusion construct with the longer linkersequence-GTGGGSGGGT-(SEQ ID NO:15). The MSP2 with the minimal linker,however, assembles into particles very similar to particles containingtwo MSP1 proteins, but which are more stable than those comprised of twoMSP1 proteins. It is understood that the best choice of the particularMSP depends on the particular protein with which is to be assembled. Ingeneral, the assembly with larger proteins or protein complexes requiresthe use of larger MSPs.

One important goal in utilizing a membrane scaffold protein (MSP) toprovide membrane proteins in general, and G-protein Coupled Receptors(GPCRs) in particular, with a suitable environment for homogeneousbiochemical assay or crystallization is to have homogeneous preparationsof particles. The engineered membrane scaffold proteins we havedescribed, including, but not limited to, truncated human apo A-I (MSP1)where the amino terminal soluble domain has been removed, deletion orinsertion mutants where one or more protein segments are removed orinserted, tandem repeats of MSP1 or deletion mutants, respectively, andany of the above materials where a histidine tag is incorporated,primarily form 8-10 nm (in diameter) particles when self-assembled withphospholipids in solution. Desirably the MSP does not include the helixH1. However, upon assembly with non-optimal stoichiometry of MSP andphospholipid, particles of other sizes may be present. While standardsize separation chromatography can be used to purify a single size classof particle, it is preferable to minimize the size distribution of theinitial reconstitution mixture of target protein, MSP and phospholipid.Engineered Nanodiscs of various sizes can be formed by appropriatechoice of the length of the membrane scaffold protein. The particle 8-10nm in diameter nominally comprises two MSP proteins.

Apolipoprotein Sequences

Sequences of several apolipoproteins, hemoglobins and myoglobins areavailable on the internet at the site of The National Center forBiotechnology Information (NCIB), National Institutes of Health. Thecoding sequences can be found on the internet and used in theconstruction of artificial MSP coding sequences or the sequences can betailored to optimize expression in the recombinant host cell of choice.There is a large body of information about codon choice andnontranslated sequences in the art. Apolipoprotein C sequences include,without limitation, bovine, XP 77416; mouse, AAH 28816; human NP 000032;and monkey, Q28995. Myoglobin sequences include, for example, those ofmouse, NP 038621; bovine, NP 776306; rat, NP 067599; and human, NP005359. Hemoglobin alpha chain sequences include human, AAH 32122 or NP000549; beta chain sequences include human, NP 000509 or P02023; rat, NP150237; mouse NP032246; bovine, NP 776342, all of which are incorporatedby reference herein. Others may be found at the NCBI website and in thescientific literature as well.

As used herein, amphiphilic and amphipathic are used synonymously inreference to membrane scaffold proteins. An amphiphilic protein or anamphiphilic helical region of a protein is one which has bothhydrophobic and hydrophilic regions.

MSP Design

The MSPs of the present invention must be amphipathic, with one part ofits structure more or less hydrophilic and facing the aqueous solventand another part more or less hydrophobic and facing the center of thehydrophobic bilayer that is to be stabilized. The elements of secondarystructure of the protein generate the hydrophilic and hydrophobicregions in three dimensional space. Examination of the basic biochemicalliterature reveals two candidate protein structures that can have thisrequired amphipathic character: the helix and the pleated sheet. Wedesigned the MSPs described herein to have a helix as the fundamentalamphipathic building block. Each MSP has an amino acid sequence whichforms amphipathic helices with more hydrophobic residues (such as A, C,F, I, L, M, V, W or Y) predominantly on one face of the helix and morepolar or charged residues (such as D, E, N, Q, S, T, H, K or R andsometimes C) on the other face of the helix. See FIG. 2 for a schematicrepresentation. In addition, each helical building block can be, but isnot necessarily, punctuated with residues such as proline (P) or glycine(G) periodically, which can introduce flexibility into the overallstructure by interrupting the general topology of the helix. In oneembodiment, these punctuations occur about every 20-25 amino acids toform “kinks” or to initiate turns to facilitate the “wrapping” of theMSP around the edge of a discoidal phospholipid bilayer. The punctuationregion (or sequence) can include from one to 10 amino acids, especially3 to 10 where there are antiparallel beta sheets in the MSP. See FIG. 2,which depicts a generalized linear amino acid sequence and a helicalwheel diagram showing the placement of predominantly hydrophobic aminoacids on one face of the helix.

We created an additional artificial variant MSP (MSP2) by designing atandem repeat of MSP1 connected by a short linker sequence to create anew molecule. This type of artificial MSP is termed a tandem repeat MSP.See FIG. 4G and SEQ ID NO:17. Relatively large quantities (tens ofmilligrams/liter cell culture) of the artificial MSPs of the presentinvention are produced in a bacterial expression system. Our constructsreduce the number of size classes that can be formed (thosecorresponding to three MSP1 molecules). As used herein a tandem repeatmembrane scaffold protein is one in which at least four helices of amembrane scaffold are repeated in linear order in a new membranescaffold protein (e.g.,H1-H2-H3-H4-H5-H6-H7-H8-spacer-H1-H2-H3-H4-H5-H6-H7-H8). Examples oftandem repeat MSPs are also given in FIGS. 4E and 4G. See also SEQ IDNO:17 and SEQ ID NO:19, among others.

Nanodiscs made with tandem repeats (two) of MSP1 sequences were larger,but less stable, than those using certain other MSP structures, at leastin some instances. Designing MSPs lacking at least one copy of H1allowed the preparation of stable Nanodiscs which are also larger insize. In particular, the absence of the first helix in the second halfof dimeric structure plays an important role in the improved results.

The complete amino acid and nucleic acid sequences for the MSP2 tandemrepeat scaffold protein is shown in Tables 7 and 8; see also SEQ IDNO:16 and SEQ ID NO:17. The MSP2 fusion protein was expressed in E. coliand purified to homogeneity using basically the same procedure asdescribed for the single MSPs. The MSP2 protein serves as an effectivescaffold protein, self-assembling with phospholipid upon removal ofsolubilizing detergent. At a lipid/dimer ratio of 200 corresponding tonominally 10 nm particles, there is the much greater monodispersivityafforded by the MSP2 protein. Importantly, the overall stability of thedisks, as monitored by chemically induced unfolding and exposure oftryptophan residues to solvent, is not altered by the fusion of themonomeric membrane scaffold proteins.

We have generated two new membrane scaffold protein dimers describedbelow and self-assembled these with phospholipids. The resultantNanodiscs have an overall Stokes diameter determined by small anglex-ray scattering of approximately 15.5 nm which corresponds to acalculated overall physical diameter of a discoid of 17 nm. These arethe largest Nanodiscs constructed to date. The modular sequences (seealso Table 19) of these new tandem repeat MSPs are as follows:

MSP2N2: HisTev-H1/2-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H2-H3-H4-H5-H6-H7-H8-H9-H10 MSP2N3:HisTev-H1/2-H2-H3-H4-H5-H6-H7-H8-H9-H10-GTREQLG-H2-H3-H4-H5-H6-H7-H8-H9-H10

Other MSPs have also been made and characterized. We have optimized theconditions of self-assembly to obtain the monodisperse nanoparticleswith MSP1E1, MSP1E2 and MSP1E3, shown that the length of the protein isthe determinant of the particle diameter, measured the stoichiometry oflipid/protein ratio in these particles and demonstrated the structuraldifference between the particles formed with lipids above or belowmelting point (270 K for POPC, 314 K for DPPC). We also prepared theseries of deletion mutant membrane scaffold proteins, in which onequarter, one half, or the whole first helix (residues 44-65) wasdeleted. Experiments with Nanodiscs formed with the truncated proteinsindicated that the first helix is of not required for the self-assemblyof these Nanodiscs. This observation is believed to explain the earlierdisagreement about the size of discoidal particles formed with apo A-1and their heterogeneity. SAXS data for these Nanodiscs formed withscaffold proteins of different sizes are consistent with the structuralmodel shown in FIG. 19. FIG. 19 also shows that H1 does not play asignificant role in the formation of the “belt” of MSP around theoutside of the Nanodisc.

In order to generate smaller belts around the bilayer structure, theoverall length of the helical building blocks can be reduced, and thepunctuations may be introduced more frequently. The exact amino acidsequence can vary in the positioning and number of the hydrophobic aminoacids within the designed linear sequence. Simple models in which eitherthe helical axis is parallel or perpendicular to the normal of theNanodisc bilayer can be generated. To generate a disk with a diameter ofroughly 10 nm, an MSP comprises about 12 to about 20 or more repeatingunits having this generalized amphipathic sequence. Preferably, thisprotein would be composed of amphipathic alpha helices each with alength of between 14 and 25 amino acids, punctuated in the linearsequence by a residue unfavorable for helix formation, such as prolineor glycine or a sequence from about 1 to 5 amino acids which does notfavor helix formation, which form small helical building blocks thatstabilize the hydrophobic core of the phospholipid bilayer. A helix ofabout 20-25 amino acids (a helical building block, in the context of thepresent application) has a height comparable to the thickness of amembrane bilayer. These small helical segments are linked together(punctuated) with from 0 to about 5 amino acid residues, especially G orP. To cover the edge of a 10 nm discoidal particle in either the “belt”model presented, one would need between 10-20 such helices, with 16being a useful number based on the simple graphic analysis of FIG. 3.Desirably, the helix contains from about 3 to about 18 amino acids perturn, and the type of helix can be an alpha, pi or 3, 10 helix, amongothers. Helices with three to sixteen, three to eight, desirably threeto four, amino acids per turn of the helix. An MSP of the presentinvention can comprise from 50 to 400 turns. Secondary structurepredictions can be determined using programs readily accessible to theart; see, for example, on the internet at the ExPASy proteomics serverof the Swiss Institute of Bioinformatics. Guidance in predictingsecondary structure is also given in publications such as Chou et al.(1974) Biochemistry 13:211, 222; Chou et al. (1978) Ann. Rev Biochem.47:251-278; Fasman (1987) Biopolymers 26(supp.):S59-S79. Where there isa dimer or higher oligomer of a protein such as a 7-TM membrane proteinor where more than one protein is to be incorporated within a singleNanodisc, for example a reductase and a cytochrome, the MSP used must becapable of forming a Nanodisc particle with a diameter greater than 9-10nm. The larger Nanodiscs are prepared using longer MSP sequences, suchas MSP1E1, MSP1E2 or MSP1E3.

In an alternative embodiment, the engineered amphiphilic MSP containregions of secondary structure in three dimensional space, such asparallel or antiparallel beta sheets, with spacer regions of appropriatelength to allow association of hydrophobic regions with a targethydrophobic target molecule which is protected from the aqueous milieu,and thus stabilized and solubilized.

Certain critical systems controlling cellular function are located inmembrane compartments. Many of these membrane protein assembliesrepresent important pharmaceutical targets that are typically difficultto isolate in soluble and active form because particular phospholipidenvironments are often essential for maintaining optimal enzymaticturnover or ligand binding activity. Several pharmacologicallysignificant examples indicate specific phospholipid requirements forindividual enzymes and receptors, which are perturbed by detergentstypically used to solubilize membrane proteins. Examples include thehuman β-adrenergic receptor that requires neutral lipids for efficientreceptor hormone response (Kirolovsky et al., 1985) and the humancytochrome P450 monooxygenase (P450) superfamily that requires severalphospholipid types for efficient drug metabolism (Imaoka et al., 1992).An inability to faithfully reconstitute the lipid requirements ofdetergent solubilized protein in purified systems can, and often does,affect the measured activity of these enzymes.

One of the most widely used alternatives for characterization of thesenative proteins involves the sub-fractionation of natural cellularmembranes and incorporation into micron-sized liposomes. However,liposomes are compromised by thermodynamic instability, sizeheterogeneity and sequestration of target membrane proteins on thesolvent-inaccessible side of the bilayer (Angrand et al., 1997; Savelliet al., 2000). Other convenient methods for obtaining large quantitiesof soluble functional membrane proteins assembled in phospholipidbilayers have not been available and, as a consequence, ourunderstanding of the numerous protein complexes functioning within cellmembranes has been hindered. In this application, we report a rapidmethod for compartmentalizing heterologously-expressed or nativemembrane proteins into stable, soluble nanometer-scale bilayerstructures which are characterized by sufficient target stability,biological activity and sufficient robustness to survive operation inhigh-throughput analyses.

The MSPs of the present invention can be used to solubilize tethered,embedded or integral membrane proteins in nanoscale structures. Atethered membrane protein is composed mostly of a single relativelysoluble globular domain external to the bilayer and a relatively simple(e.g., a single membrane-spanning or membrane-inserting domain) whichanchors this simple globular domain to the membrane bilayer. Theglobular domain, in nature, can be extracellular or cytoplasmic inorientation.

Tethered membrane proteins are exemplified by NADPH-cytochrome P450reductases (e.g., from rat liver endoplasmic reticulum or from insect)and cytochrome b5. NADPH-Cytochrome P450 reductase is a membrane proteinfound in the endoplasmic reticulum. It catalyzes pyridine nucleotidedehydration and electron transfer to membrane bound cytochrome P450s.Isozymes of similar structure are found in humans, plants, othermammals, insects etc

Cytochrome b5 is a membrane-anchored (tethered) heme protein having asingle membrane anchor domain that penetrates the membrane bilayer.Cytochrome b5 solubilized from its native membrane exists as largeaggregates in the absence of detergent and appears as a smear ratherthan a discrete band on native polyacrylamide gel electrophoresis(PAGE). Formation of Nanodiscs through the self-assembly process usingMSPs taught in our invention,, wherein cytochrome b5 is added to thepreparation of MSP and phospholipid results in incorporation ofcytochrome b5 into disk structures. The disk complexes containingcytochrome b5 can be chromatographically separated and purified fromundesired aggregated material. The optical absorption properties of theheme chromophore of the purified material show that the heme active siteis in a native conformation. Tethered membrane proteins can beincorporated into Nanodiscs either during disc formation, or they canassociate with preformed Nanodiscs.

Embedded membrane proteins, as defined herein, are those which include amembrane anchoring segment of the polypeptide, but which also havegroupings of hydrophobic amino acids on the surface of the protein,which hydrophobic domains are embedded within the membrane bilayer.Examples of embedded membrane proteins include, without limitation, theinterferon receptor superfamily, the nerve growth factor/tumor and thenecrosis factor receptor superfamily as well as the cytochrome P450proteins.

Tissue factor (TF), or thromboplastin, is a 30,000 Da type-I membraneprotein critical to initiation of the blood coagulation cascade. Thismembrane-bound protein acts as an activation cofactor for factor VII,the soluble serine protease which carries out the first enzymatic stepin blood coagulation. Expression of tissue factor is limited to cellsthat are not in direct contact with blood plasma, which cells form a“hemostatic envelope.” The TF:VII complex must be assembled on amembrane surface to exhibit high activity, and optimal activity is seenonly when the membrane contains phospholipids with negatively chargedheadgroups.

Another integral membrane protein which has been incorporated intoNanodiscs is a bacterial aspartate receptor. In E. coli and Salmonellathe chemoreceptors Tsr and Tar mediate taxis towards serine andaspartate, respectively, mediated through stereospecific binding ofthose amino acids. In E. coli the Tar receptor protein mediates taxistowards maltose via recognition of a ligand-occupied solublemaltose-binding protein. Membranes from E. coli containingover-expressed Tar protein (provided by Gerald Hazelbauer, University ofMissouri, Columbia, Mo.) were solubilized with CHAPS detergent and mixedwith the scaffold protein MSP1T2. Detergent was removed by adsorption(using Biobead treatment as described herein below). The Tar receptorwas incorporated into Nanodiscs, which were then purified by Ni-affinitycolumn chromatography and analyzed by HPLC size-exclusionchromatography. Incorporation of the target was verified by SDS PAGE.

Examples of embedded cytochrome P450 membrane proteins include, withoutlimitation, cytochrome P450 2B4 from rabbit liver microsomes, cytochromeP450 3A4 from human liver microsomes, cytochrome P450 6B1 from insectfat bodies, and cytochrome P450 86A1, 73A5 and 86A8 from plants thecytochromes P450 are a superfamily of enzymes that are found in allforms of life. One role of many P450s is to detoxify xenobiotics; forinstance, human liver P450s detoxify most endogenous and exogenouscompounds, and these enzymes determine the mean plasma lifetime of alldrugs ingested. One of the most widely studied human liver cytochromeP450s is cytochrome P450 3A4 (CYP 3A4). This membrane bound P450 is themost highly expressed P450 in human liver and is responsible formetabolizing almost 50% of all pharmaceuticals (Guengerich, F. P.,Cytochrome P450. Cytochrome P450, ed. P. R. Ortiz de Montellano, 1995,New York: Plenum Press. 473-535). In order to demonstrate the utility ofNanodisc technology for the study of the cytochrome P450, weincorporated CYP 3A4 into MSP supported nanobilayer discs. Furtherevidence from size separation chromatography and PAGE analysis supportsthe conclusion of incorporation of CYP 3A4 into Nanodiscs.

Cytochrome P450 6B1 (CYP 6B1) is a member of the large cytochrome P450monooxygenase protein superfamily, and it is another example of anembedded membrane protein. CYP 6B1 has been isolated from Papiliopolyxenes, the black swallow tail, which feeds exclusively on plantsproducing furanocoumarins, plant psoralen derivatives that arephototoxic to most organisms. CYP 6B1 catalyzes the detoxification offuranocoumarins by what is believed to be an epoxidation reaction (Ma etal. (1994)).

Integral membrane proteins have predominant and critical regions ofstructure located within the membrane bilayer. Alternatively, there canbe relatively large soluble domains on both sides of the bilayer whichare linked by one or more passes of the primary sequence through thehydrophobic bilayer core, especially cytokine-type molecules andreceptors, which have simple one-pass connectivity but with solubledomains on both sides of the bilayer. As used herein, integral membraneproteins are exemplified by the general class of proteins in which thereone or more helical segment in the membrane bilayer, including but notlimited to the well known 7 helix transmembrane proteins (e.g., GPCRs).

We have shown that MSP1, MSP2, MSP1E1, MSP1E2, MSP1E3 and MSP2 assemblewith bacteriorhodopsin. From the initial reconstitution mixture, twobacteriorhodopsin-containing species are observed when particles areformed with MSP1 or MSP2 in the absence of added phospholipid. MSP isabsolutely required for the solubilization of bacteriorhodopsin to formthese species because omission of an MSP from the formation mixtureresults in large non-specific bacteriorhodopsin aggregates that elute inthe void volume of the gel filtration column. The majority ofbacteriorhodopsin appeared solubilized in the presence of MSPs.

An especially valuable advantage of the MSP-containing nanoparticles ofthe present invention as a means to solubilize hydrophobic or partiallyhydrophobic “target” proteins is that the protein incorporated into thenanoparticle has a naturalistic presentation. Native target proteinconformation is maintained, the native target protein-membraneinteraction and topology are preserved, the target protein is maintainedin a native-like environment, thereby increasing the stability of thetarget protein to inactivation and denaturation, and the topology of thetarget protein is maintained relative to the membrane. The maintenanceof target protein topology relative to the membrane is especiallyimportant for screening targets for cell-cell or cell-virus interaction,elicitation of ligand or antibody binding to extra-membrane regions ofthe target protein or delivery of the target protein through specifictrafficking pathways.

Incorporation from Membranes and Membrane Fragments

We have demonstrated that membranes or membrane fragments containingtheir natural repertoire of membrane proteins and lipids can beincorporated into Nanodiscs comprising MSPs. This can be effecteddirectly without pre-purification or solubilization of the membraneprotein populations. A particularly important embodiment is the use ofthis technology in a variety of commonly used heterologous expressionsystems for membrane proteins. These include, but are not limited to,insect cells, yeast cells, mammalian cells such as HEK cells, Vero cellsand CHO cells, and bacterial cells. Virus envelope proteins or cellmembranes of pathogens (e.g., bacteria), either of which can containmultiple copies of antigenic proteins or other molecules, can also beused. A specifically exemplified embodiment is the use of the commoninsect cell-baculovirus expression system. We used a commerciallyavailable Sf9 insect cell line co-infected such that a microsomalpreparation containing over-expressed insect CYP6B1 and anover-expressed insect NADPH cytochrome P450 reductase was produced.Hence, we not only demonstrated that MSP Nanodiscs can be used toincorporate another cytochrome P450 system into soluble monodisperseparticles, but also that the source of this P450 could be the wholemembranes from the Sf9 cell line that was infected with a baculoviruscarrying a cloned CYP6B1 gene.

The Nanodiscs generated by the procedure described herein contain thefatty acids and phospholipids from the original native membrane startingmaterial and therefore provide a reliable in vitro environment in whichto assay any membrane-bound enzyme or receptor of interest. Thus,MSP-supported Nanodiscs can be used in high-throughput screeningventures such as the identification of ligands for membrane-associatedproteins, for example, using combinatorial libraries of peptides,proteins or chemical compounds, and for the identification of newpharmaceuticals. Additionally, the simple procedure of incorporationinto Nanodiscs can be used to generate samples for structuredetermination using x-ray crystallography or NMR spectroscopy. Aparticular advantage of the Nanodisc system over alternative methods formembrane protein solubilization is the increase in sensitivity ofoptical measurements due to a significant decrease in light scatteringof the particles. The methods of the present invention can be extendedto any other source of membrane fragments containing target proteins ofinterest, such as any yeast, insect, bacterial or mammalian cell culturesystem or expression system.

High Throughput Screening

An important utility of the Nanodisc technology of the present inventionis in high throughput screening for enzymatic or ligand bindingactivity. In many such systems, it is advantageous to have more than onetarget membrane protein incorporated into the Nanodiscs, for example,the electron transfer partner needed for P450 monooxygenase catalysis orthe corresponding G-protein incorporated with a G-protein coupledreceptor.

In order to demonstrate the utility of the MSP Nanodisc technology inthese situations, we successfully incorporated the NADPH cytochrome P450reductase and a cytochrome P450 6B1 into Nanodiscs. As demonstratedherein, each target membrane protein can be individually incorporatedinto Nanodiscs using MSPs or they can be incorporated in combinations.The endogenous relative amount of cytochrome P450 to reductase is about10-20 P450 molecules per reductase molecule (Feyereisen, R. (1999) Ann.Rev. Entomol. 44, 501-533). To obtain activity of CYP6B1 afterreconstitution into disks, an excess amount of reductase can be added tothe reconstitution mixture.

BR, an integral membrane protein, has been incorporated into the MSPNanodiscs as described herein, and we have also used a commerciallyavailable insect cell expression system that provides a membranefraction hosting the G-protein coupled receptor human for 5-HT-1A(serotonin). The ligand binding activity documented for 5-HT-1Aincorporation into Nanodiscs proves that the protein is in the activeconformation in the Nanodiscs of the present invention. Subsequentexperiments show that the beta-2 adrenergic receptor, the dopamine D2and D1 receptors and the cytokine receptors CXCR4 and CCR5, all of whichbelong to the 7-transmembrane protein family and G-protein coupledreceptor type, are easily incorporated into Nanodiscs by the methods ofthe present invention.

Other examples of membrane proteins and membrane protein complexes whichhave successfully been incorporated into Nanodiscs include cytochromeP450 reductases from rat, insects and plants, a bacteriorhodopsintrimer, a photosynthetic reaction center complex, a twenty-sixtransmembrane domain Escherichia coli transhydrogenase and integrin.

Stoichiometry of protein and lipid is an important factor in theformation of monodisperse discs with all MSPs. The set of extended MSPsshows that there is a well-defined optimum of lipid/protein ratio, whichis crucial for quantitative assembly of monodisperse discoidalparticles. In addition, the stoichiometric ratio is determined by theMSP length, because of the well-defined topology of discoidal structurewith a cylindrical lipid bilayer surrounded by the scaffold protein(FIG. 19), which determines the lipid/protein ratio. The lipid/proteinstoichiometry ratios for discs of different sizes were calculated asshown at FIG. 19 and tested experimentally. Concentrations of lipidswere measured using tritiated lipids of the same chemical structure andscintillation counting of the column fractions, as described (Bayburt etal. (2002) supra). Concentrations of scaffold proteins were determinedspectrophotometrically (Jones et al. (1990) J. Biol. Chem.274:22123-22129), using the molar absorption coefficients calculated forthe known amino acid sequences according to the modified method ofGill-von Hippel, as described in Pace et al. (1995) Protein Science4:2411-2423. All measurements were done on the narrow fractions afterseparation of the assembled lipid-protein particles by HPLC (MilleniumSystem, Waters, Milford, Mass.) on the calibrated Superdex 200 (sizeexclusion chromatography) column.

Small angle X-ray scattering (SAXS) was measured at an ambienttemperature of 295 K at the vacuum chamber with 1500 mm distance fromthe sample to the 2D detector at the photon energy 15 keV (wavelength0.826 A). The solutions of the Nanodiscs were sealed in glasscapillaries with a diameter of 1.5 mm and placed on the holder in thesample chamber, together with calibrant (Ag behenate, spacing 58.38 Å,)and reference buffer solvents. The raw data were processed using theprogram FIT2D (Hammersley, A. P. (1998) ESRF Internal Report,ESRF98HA01T, FITD2D V9. 129 Reference Manual V3.1; Hammersley et al.(1996) High Pressure Research 14:235-248) to give the scattering curvesin the form 1 g(I/I₀) vs. Q=4π sin(q)/2. Analysis of SAXS data was withthe program CRYSOL (Svergun et al. (1995) J. Appl. Cryst. 28:768-773)and home written modeling and fitting subroutines using MATLAB(MathWorks, Natick, Mass.). The fitting program is based on the Debyeequation and modeling of the nanoparticle by close packed sphericalbeads with different contrast, as it is done in other popular programsCRYSOL, DUMMIN, SAXS3D and DALAI_GA, reviewed in Takahasi et al. (2003)J. Appl. Cryst. 36:549-552; Koch et al. (2003) Quart. Rev. Biophys.36:147-227. The models for fitting were constructed using theinformation on size and composition of Nanodiscs obtained by othermethods described in this paper. The initial estimate for the scatteringcontrast, i.e. the difference between the electron density for water,0.334 e/Å⁻³, and the average electron density for the methylene groupsin the central part of bilayer, lipid acyl chains, lipid polar headgroups (Wiener et al (1989) Biophys. J. 55:315-325), and scaffoldprotein at the circumference of the particle (Svergun et al. (1998)Proc. Natl. Acad. Sci. USA 95:2267-2272), was assigned to each beadrepresenting a correspondent phase. Experimental curves for each type ofNanodisc were fitted using five parameters, four electron densities andthe radius of the disc. No attempt to made to introduce size or shapeheterogeneity into the fitting. Modeling of such heterogeneity within 5%(as suggested by other experimental data) did not give a largedifference in the calculated scattering curves, more significantvariance in size and shape resulted in the loss of observed features onthe scattering curves and gave considerably worse fits.

MSPs have been engineered to minimize the variability in the structureof the discoidal phospholipid bilayer entities, provide greaterstructural stability and increased size homogeneity of the diskstructures, and incorporate useful functionalities such as peptide tagsfor purification and physical manipulation of disks. Such oligopeptidetags which can be fused to a protein of interest (by molecularbiological or chemical methods) include, without limitation, strep-tag(Sigma-Genosys, The Woodlands, Tex.) which directs binding tostreptavidin or its derivative streptactin (Sigma-Genosys); aglutathione-S-transferase gene fusion system which directs binding toglutathione coupled to a solid support (Amersham Pharmacia Biotech,Uppsala, Sweden); a calmodulin-binding peptide fusion system whichallows purification using a calmodulin resin (Stratagene, La Jolla,Calif.); a maltose binding protein fusion system allowing binding to anamylose resin (New England Biolabs, Beverly, Mass.); and theoligo-histidine fusion peptide system which allows purification using aNi²⁺-NTA column (Qiagen, Valencia, Calif.).

Disk homogeneity is necessary for efficient incorporation of singlemembrane proteins or single membrane protein complexes into a singlesize class of disk. The parent molecule, apo A-I, has functions beyonddisk structure stabilization (Forte et al., 1971; Holvoet et al., 1995;Fidge, 1999). These functional regions are unnecessary and oftendeleterious in the artificial bilayer systems of the present invention.

Secondary structure prediction allows assessment of structural featuresof the scaffold protein. The apo A-I structure consists of mostly helix,sometimes punctuated by proline or glycine residues in the repeatsequences. Eight to nine helices are believed to associate with lipid inthe form of disks. The N-terminal “GLOB” region (SEQ ID NO:89) of apoA-I is predicted to be more globular in character. This portion of themolecule has been removed to produce the engineered MSP1. An MSP thatproduces disk assemblies with high monodispersity is desirable. Toascertain the roles of half repeats and to further characterize andoptimize the MSP structure and function, mutagenesis was used togenerate variants as described herein below. See Tables 2-21 below.

Hydrophobic or partially hydrophobic receptors incorporated into MSPdisks are useful in structural, biochemical and pharmaceutical research.Membrane protein study was previously limited to insoluble membranedispersions, detergent micelles, and liposomes. Purified systems forbiochemical and physical study require stability, which may or may notbe obtainable with detergents. Detergent micelles are dynamic andundergo structural fluctuations that promote subunit dissociation andpresent difficulties in the handling of proteins in dilute solution. MSPnanobilayers (Nanodiscs) are more robust structurally, having aphospholipid bilayer mimetic domain of discrete size and composition,and greater stability and smaller surface area than unilamellarliposomes. The particles of the present invention are stable in size,conformation and biological activity for at least a month at 4° C.

Surface Technology

The MSPs of the present invention, when formulated into Nanodiscs, canbe used in analyses in surface technology such as biosensor chips forhigh throughput screening or solid phase assay techniques, including butnot limited to multiwell plates made, for example, of polystyrene. Wherethe MSP comprises a His tag, the Nanodiscs can be bound to animmobilized metal, for example divalent nickel cation. Our work on diskscaffolds has also involved surface-associated assemblies.

For instance, the surface plasmon resonance (SPR) biosensor utilizes anapproximately 50 nm gold film on an optical component to couple surfaceplasmons to a dielectric component (sample) at the surface of the goldfilm. MSP-stabilized bilayers can be attached to the surface for use asa biomimetic layer containing proteins or other targets of interest byengineering cysteines into the MSP (FIG. 7A). The use of thiols is wellknown for attaching molecules to gold surfaces. Based on the belt model,cysteine residues can be placed along the polar side of the amphipathichelix axis, provided that a cysteine residue is not positioned at thehelix-helix interface. In cases wherein the MSP is so engineered,multiple cysteine residues can form disulfide-linked dimers (Segrest etal., 1999). An alternative is to introduce cysteines within a flexibleN- or C-terminal linker. Such a construct is, in theory, capable ofassociating the belt (or the picket fence) model of disk to a goldsurface. Alternatively, thiol lipids can be incorporated within thebilayer domain. Methodologies which utilize differences in opticalrefractive index with layered structures, such as total internalreflection spectroscopy, resonant mirrors, optical diffraction gratingengineered on optical surfaces, and the like, can be likewise utilizedby direct extrapolation. In addition to SPR, surface-associated disks ongold can be used in STM and electrochemical studies, for example, suchas with membrane associated redox proteins, e.g. cytochrome P450 and itsflavoprotein, as well as ion channels.

SPR data can also be obtained from measurements made using a thin filmof dielectric such as silicon dioxide applied over the metal filmnormally used as the substrate in SPR. This variation of the techniquehas been termed coupled plasmon waveguide resonance (CPWR) (Salamon etal., 1997a). Because silica can be used as the active surface in theseplasmon resonance experiments, the process of producing a self-assembledbilayer can be adapted according to the procedures used to producesurfaces on mica or other silicon oxide surfaces. This has the addedadvantage of making the conditions used for the SPR experiments directlycomparable to those used for AFM experiments. The CPWR technique caneasily be performed on an SPR instrument by simply adding the silicacoating to the metal film slides that are presently used for SPRspectroscopy.

MSPs with available cysteine groups also enable specific labeling withchemically reactive groups or affinity tags for immobilization in gelmatrices. Hydrogels with reactive coupling groups are useful forimmobilizing proteins for SPR measurements. In a hydrogel configuration,the disk serves as a carrier for bilayer-embedded membrane proteins in amonodisperse form with both intra- and extracellular domains availablefor ligand binding. We have already demonstrated that disks containing aHis tag bind to a metal chelate matrix, which can be used to immobilizeNanodiscs containing a His-tagged MSP. His tag vectors are commerciallyavailable (e.g., from Qiagen, Valencia, Calif.) and are described inU.S. Pat. Nos. 5,284,933 and 5,130,663. Other tag peptide sequencesknown to the art, including but not limited to, Flag tag (flagellarantigen) or Step tag (streptavidin binding), can be engineered into theMSP by molecular biological methods. Besides mediating attachment to asupport of choice, the tag sequences can facilitate purification of theMSPs or of Nanodiscs containing them. Nanodiscs can also be used inpreparing affinity matrices for bioseparation processes and measurementsof ligand affinities. The particles produced by the methods of thepresent invention are useful for drug discovery, structure/functioncorrelation, and structure determination of membrane proteins.

Membrane Protein Structure and Function Analysis

Structure determination of membrane proteins has been limited by theabilities to produce large amounts of membrane proteins and tocrystallize these proteins. Nanodiscs and MSPs are useful as carriersfor membrane protein stabilization and expression. MSP can serve tosolubilize membrane proteins for crystallization in lieu of detergents.Indeed, where the lipid bound form of MSP is structurally stable andrigid, crystallization can be enhanced by introduction of crystalcontacts through the MSP. We have demonstrated that MSP1, the extendedforms of MSP1, and MSP2 or other tandem repeat MSPs can be used tosolubilize BR from purple membranes in the presence and absence ofexogenous lipid.

Fusion constructs with a membrane (or other) protein and an MSP regioncan be expressed in Escherichia coli using any of a number of art-knownvectors to produce a stable and soluble form of the membrane proteinthat contains a membrane anchor in large quantity. The excitingdiscovery that MSP solubilizes BR in the absence of added phospholipidallows the use of the artificial MSP to stabilize membrane proteins inthe absence of detergents or lipid additives. The (artificial) MSPsdisclosed herein can be used in solubilization of other membraneproteins including, but not limited to, cytochrome P450, cytochrome P450reductase, and the 5-HT-1A receptor, as well as othermembrane-associated receptor proteins and enzymes.

Signal transducing elements occur across membranes, while vesiclesrender one side of membrane inaccessible to hydrophilic reagents andeffector proteins. A specific embodiment of the present invention usesdisks to solubilize and stabilize pharmaceutical targets such as GPCRs,ion channels, receptor kinases, and phosphatases in a naturalisticpresentation. We have incorporated proteins with multiple membranespanning domains into the disks of the present invention, with a focuson GPCRs. We had successfully incorporated the model serpentine membraneprotein, bacteriorhodopsin, into Nanodiscs. Bacteriorhodopsin is a modelfor GPCRs, which are current targets for drug discovery. Currently, over1000 probable G-protein receptors from various organisms have beencloned and many of the so-called “orphan” receptors await identificationof natural (or synthetic) ligands. Ligand classes include peptidehormones, neurotransmitters, eicosanoids, lipids, calcium, nucleotides,and biogenic amines. GPCRs are believed be targets for more than half ofcurrently marketed pharmaceuticals. This structural class of membraneproteins can readily be incorporated into Nanodiscs when contacted withMSPs as pre-solubilized proteins or as membrane-associated proteins.G-protein coupled receptors inserted into Nanodiscs are completelyfunctional in this trans-membrane signaling process. Structuralcharacterization of the reconstituted receptors is performed usingchemical analysis, spectroscopy and atomic force microscopy.

Cytochrome proteins and reductases can be derived from plant, insect,mammalian, avian or other sources. Specific examples include, insectcytochrome P450 reductase and cytochrome P450 CYP6B1 and plantcytochrome P450 CYP7B12, CYP7B13, CYP73A5, CYP86A1, CYP86A2, CYP86A4,CYP86A7 or CYP86A8. “Derived from” can mean that the target protein ispresent in a natural (native) membrane when contacted with MSP toproduce Nanodiscs, or the target protein can be isolated, purified orpresolublized, or the target protein can be associated with themembranes of cells in which it is recombinantly produced.

GPCRs which can be solubilized in nanoscale phospholipid bilayersinclude the Class A (Rhodopsin-like ) GPCRs which bind amines, peptides,hormone proteins, rhodopsin, olfactory prostanoid, nucleotide-likecompounds, cannabinoids, platelet activating factor,gonadotropin-releasing hormone, thyrotropin-releasing hormone andsecretagogue, melatonin and lysosphingolipid and lysophosphatidic acid(LPA), among other compounds. GPCRs with amine ligands include, withoutlimitation, acetylcholine or muscarinic, adrenoceptors, dopamine,histamine, serotonin or octopamine receptors; peptide ligands include,but are not limited to, angiotensin, bombesin, bradykinin,anaphylatoxin, Fmet-leu-phe, interleukin-8, chemokine, cholecystokinin,endothelin, melanocortin, neuropeptide Y, neurotensin, opioid,somatostatin, tachykinin, thrombin vasopressin-like, galanin, proteinaseactivated, orexin and neuropeptide FF, adrenomedullin (G10D),GPR37/endothelin B-like, chemokine receptor-like and neuromedin U.

Other exemplary proteins include mammalian, especially human, CCR5 andCXCR4 chemokine receptors. These were incorporated into Nanodiscs bycontacting membranes containing native or recombinant protein. Thenative protein conformation is maintained, as evidenced by the reactionof the CCR5-containing and CXCR4-containing Nanodiscs with CCR5- andCXCR4-specific antibodies. Nanodiscs containing the human bet-2adrenergic receptor have also been made.

Ligands of other specific GPCRs include hormone proteins, rhodopsin,olfactory compounds, prostanoid, nucleotide-like (adenosine,purinoceptors), cannabinoid, platelet activating factor,gonadotropin-releasing hormone, thyrotropin-releasing hormone andsecretagogue, melatonin and lysosphingolipid and LPA, among others.Class B secretin-like GPCRs include, without limitation, those whichbind calcitonin, corticotropin releasing factor, gastric inhibitorypeptide, glucagon, growth hormone-releasing hormone, parathyroidhormone, pituitary adenylate cyclase activating polypeptide (PACAP),secretin, vasoactive intestinal polypeptide, diuretic hormone, EMR1 andlatrophilin. Class C metabotropic glutamate receptors include thosewhich bind metabotropic glutamate, extracellular calcium-sensingreceptors or GABA-B receptors, among others. “Orphan” receptors whoseligands are not yet known are also potential targets of assays of thepresent invention.

In the assays of the present invention which demonstrate binding of aparticular ligand or which are used to identify inhibitors orcompetitors of ligand binding to an MSP-supported GPCR, a variety ofdetectable moieties (labels) can be incorporated within the ligandmolecule (such as radioactive isotope, e.g., ³H, ¹⁴C, ³⁵S, ³²P, ¹²⁵I,¹³¹I, fluorescent compounds, luminescent compounds, etc.) can beattached to the ligand molecule provided that binding to the cognatereceptor is not significantly reduced due to the label.

Scanning Probe Microscopy

An important technique used in the characterization of disk structuresand associated proteins is scanning probe microscopy (SPM). SPM is anumbrella term for any microscope that utilizes the scanning principlesfirst pioneered in the scanning tunneling microscope (STM), but thesemicroscopes can vary so greatly they are best discussed in terms oftheir guiding central principle. The technology has been used in theanalysis of biological membranes and their associated proteins, bilayerstructures and incorporated membrane proteins surfaces. SPM combinesindependent mobility in all three spatial directions (scanning) with adetection system capable of detecting some characteristic of the surface(probing). The various surface characteristics that can be probed(conductivity, surface forces, compressibility, capacitance, magnetic,fluorescence emission) demonstrate the wealth of information that can beobtained. The excellent z-axis sensitivity of atomic force microscopymakes the presence of proteins binding to an rHDL monolayer or inNanodiscs easily detectable (Bayburt et al., 1998). Precise heightmeasurements are possible with AFM, and membrane protein heightmeasurements obtained by modulating the force of the AFM probe onvarious Nanodisc assemblies (Bayburt et al., 2000). The surfaceassociation of disks formed from MSPs allows direct investigation of thebiophysical properties of single membrane proteins incorporated intophospholipid bilayers on surfaces by SPM. The ability to attach disks toatomically flat conductive surfaces (such as gold or silica) isnecessary for scanning tunneling microscopy (STM). Without wishing to bebound by theory, it is believed that tunneling through a redox-activesystem can be used to probe the functional state of an enzyme (Friis etal., 1999; Mukhopadhyay et al., 2000). These two techniques providecomplementary data and can be used in concert to study events occurringat the bilayer/solution interface. The ability to place disks on a goldsurface also allows the use of surface plasmon resonance (SPR).Insertion of membrane proteins into such artificial lipid bilayers, ortheir interaction with surface-associated proteins can be detected andquantified by SPR.

Other useful solid surfaces onto which Nanodiscs can be bound include,without limitation, quartz, silica, silicon, silicon oxide, siliconnitride, polystyrene, plastic and resins.

Disc Stability and Size Dispersion

Measurements of disk stabilities and determination of size dispersionamong classes are necessary to evaluate the constructs and Nanodiscs.Gel filtration and native gel electrophoresis are used to separate andquantitate sizes of particles. Spectroscopy is used to quantitatesecondary structure (CD) and lipid association (fluorescence)characteristics of the engineered MSPs, including stabilities based onthermal and chemical denaturation. Compositions and stoichiometries ofcomponents in disks can be quantitated by traditional methods, usingradioactive or fluorescent labels, mass spectrometry, etc. of proteinand lipid components.

Advances in the incorporation of fluorophores into the lipid bilayer ofNanodiscs have been accomplished. Such experiments provide importantinformation for the incorporation of small molecules into Nanodiscs fortherapeutic use and in the generation of labeled structures for tissuelocalization and ADME/toxicology studies. Fluorescence is one of themost widely used techniques to track proteins and to analyze proteinbinding events. Nanodiscs can be prepared to contain lipophilicfluorescent dyes and to label proteins. Several different fluorophoreshave been incorporated into the lipid bilayer of Nanodiscs during orafter self-assembly. Due to the small size of the lipid bilayer ofNanodiscs (˜8 nm in diameter) the dye is held within a few nanometers ofa protein incorporated into the bilayer. In addition, the protein-to-dyestoichiometry can be strictly controlled. This methodology allows adesired number of dyes to label a protein without directly attaching thedyes through mutations or other invasive or potentially destructivetechniques.

In related experiments, numerous fluorescein-labeled lipids were used inthe formation of Nanodiscs. Results have suggested that as many as 30 to40 small molecule organic molecules can be incorporated into a singleNanodiscs without perturbing the discoidal bilayer structure, asmonitored hydromatically. These highly fluorescent Nanodiscs are usefulin optical sensing and sorting applications, including use inmicrofluidic arrays and on-chip analytical systems for diagnostics.

As an example of this technology, Nanodiscs were assembled using DPPCdoped with DHPE-fluorescein lipids and MSP1. Lipid mixtures containing10 and 20% DHPE-fluorescein yielded Nanodiscs as shown by size exclusionchromatography. These percentages correspond to 16 and 32 fluorescentlylabeled lipids per Nanodisc which have been shown to contain 160 DPPCswhen assembled with MSP1. A variety of lipophilic fluorophores have beenincorporated into Nanodiscs. These include a lipophilic derivative offluorescein, the lipid phase state marker laurdan and a derivative ofhydroxycoumarin, a pH sensitive probe. These fluorophores have beenincorporated into Nanodiscs both during and after the assembly process.Laurdan has been incorporated into Nanodiscs containing DPPC and DMPC.All of the fluorophores have been incorporated into Nanodiscs containingDMPC as well as into Nanodiscs which have been preassembled toincorporate an integral membrane protein target.

Incorporation of Hydrophobic or Amphipathic Compounds

Hydrophobic or amphipathic organic compounds, for example fluorescentand/or lipophilic dyes such as those used to probe membrane structure,can be readily incorporated into Nanodiscs in one of two ways. Mostcommonly, such a compound can be added to the detergent solubilizedmixture. The compound of interest is then assembled naturally into thefinal structure during the Nanodisc assembly which is initiated bydetergent removal. Alternately, these compounds can be incorporated intopre-formed Nanodiscs by simple incubation. In this case, there is anexpected more facile incorporation into a fluid phospholipid state whichis determined by the incubation temperature relative to the phasetransition temperature of the phospholipid mixture. However, strongpartitioning of such compounds into the hydrophobic bilayer structureallows successful incorporation even at room temperature (about 25° C.)with DPPC (phase transition temperature about 42° C.). Lipophilic dyeswhich partition into Nanodiscs can include, without limitation,diphenylhexatriene, octyldecylindocarbocyanine (Dil), C1-BODIPY 500/510,dihexadecanoylglycerophosphoethanolamine fluorescein.

Hydrophobic or partially hydrophobic imaging agents, therapeutic and/orcosmetically active molecules and the like can also be incorporatedusing the same or similar protocols.

Atomic Force Microscopy

AFM is used to provide molecular resolution data on the structuralorganization of the lipid and protein components of the Nanodiscs of thepresent invention. This technique can be used in air, vacuum, and underaqueous and non-aqueous fluids. The latter capability has made it themost important scanning probe technique in the biological sciences. TheAFM is a very versatile instrument as it is capable of acquiring imagesand other forms of force data in contact, tapping, phase, and lateralforce modes (Sarid, 1994). These scanning modes are available on theDigital Instruments Multimode Scanning Probe Microscope (DigitalInstruments, Plainview, N.Y.), and they have been successfully used toimage rHDL and proteins associated with Nanodiscs both with and withoutincorporated proteins. This instrument can also be used in STM andelectrochemical modes to study characteristics of gold-associatedNanodiscs and incorporated redox proteins.

Modifications of MSP primary structure can generate alternative and moreeffective and stable membrane scaffold proteins. For instance, we havedeleted and/or duplicated helical regions of MSP1 to produce novelartificial membrane scaffold proteins. See Table 21 herein below forexamples of such membrane scaffold protein constructs.

Careful attention to the concentrations of MSP in the reconstitutionmixture is necessary to insure homogeneity with respect to the sizes ofNanodiscs produced. The optimal phospholipid to MSP ratio depends on theoverall size Nanodisc generated, which is in turn determined by theoverall length of the encircling membrane scaffold protein. For example,the MSP1 scaffold protein self assembles to form a nominally 9.7 nmdiameter disc with 163 DPPC phospholipid (PL) molecules incorporated perNanodisc (81.6 per MSP1). For Nanodiscs which are engineered to belarger by adding additional helical segments within the MSP, morephospholipids (PL) are enclosed. MSPE1 with an additional 22-mer helixgenerates particles of diameter 10.4 nm and 105.7 PL per MSP1E1. Withtwo 22-mer helices inserted into the MSP, a Nanodisc of diameter 11.1 nmis generated with 138.2 PL molecules per MSP1E2. With three 22-merhelices added, a 12 nm particle is produced with 176.6 DPPC moleculesper resulting Nanodisc.

We have studied the lipid composition of Nanodiscs formed with naturalcell membranes. The successful application of MSP technology to theassembly of nanobilayers from natural biological membranes provides aunique opportunity for the direct isolation of membrane proteins fromcells and their solubilization and dispersal into a system that closelymimics the native cell environment. To further clarify the extent towhich the phospholipid content of the isolated Nanodiscs mimics that ofthe original Sf9 microsomal membranes, nickel affinity-purifiednanostructures assembled with Sf9 microsomal membranes were analyzed bythin-layer chromatography. Comparisons of these Nanodisc phospholipidpopulations with the major phospholipid types found in insect cellmembranes, which are phosphatidylcholine, phosphatidylinositol, andphosphatidylethanolamine (Marheineke et al., 1998) (FIG. 17), clearlyindicate that the phospholipid composition of endogenous Sf9 microsomalmembranes is preserved in assembled Nanodiscs.

Functional Proteomics

To adapt MSP technology to a format compatible with a functionalproteomic analysis of heterologously-expressed membrane proteins,membranes from Sf9 cells overexpressing CYP6B1 were completelysolubilized with detergent in the presence of the engineered membranescaffold protein MSP1. Removal of the detergent (using Biobeads®)initiated self-assembly, allowing for the incorporation of the membraneprotein population into MSP-supported phospholipid nanobilayers, asoutlined in FIG. 14. The MSP1-containing particles were subsequentlyisolated using a nickel-chelating resin to bind the His6-tag on theN-terminus of the scaffold protein. Analysis of the affinity-purifiedsoluble nanobilayers by denaturing polyacrylamide gel electrophoresisconfirmed the presence of the CYP6B1 target protein as well as an arrayof endogenous proteins present in the original Sf9 cell membranes (FIG.15). The nickel affinity-purified sample was fractionated by sizeexclusion chromatography (FIG. 16A) and analyzed by absorbance at 417 nmto identify a 10 nm fraction containing over 90% of the solubilizedheme-containing target protein.

Size exclusion chromatography of CYP6B1-expressing Sf9 cell membranestreated and fractionated in the absence of the membrane scaffold proteinshows that the target elutes as large, non-specific aggregates (FIG.16A, dotted line). The homogeneity of the MSP1-supported Nanodiscsgenerated is dependent on the identity of lipid and its ratio of lipidto the amount of MSP used in the reconstitution procedure (Bayburt etal., 2002) supra). Our analysis of MSP disks assembled with the naturallipid pool from Sf9 insect cell membranes indicates other sizepopulations in the initial nickel affinity-purified Nanodiscs (FIG.16A). These variations are due to the difficulty in determining a priorithe precise concentration of MSP protein ideally matched to the lipidcomposition in membrane preparations expressing variable amounts of theheterologous P450 protein and to the significant size distribution ofthe endogenous membrane proteins that are also assembled intonanostructures in this process. These other size classes representnon-specific aggregates that are easily separated from the about 10 nmdiameter nanobilayer assemblies. Size-fractionated populations ofNanodiscs containing the P450 target protein are uniform and stablethrough re-fractionation on a sizing column, such as Superdex™ 200. Thefinal CYP6B1-containing population displays a stoichiometry ofapproximately one CYP6B1 protein per 10 Nanodiscs (FIG. 16B).

We have examined the integrity of the membrane protein assembled intoNanodiscs. CYP6B1-containing nanostructures were assayed by reduction ofthe iron and binding of carbon monoxide (CO), which monitors via anabsorbance maximum at 450 nm the quantity of protein that is intact andcorrectly configured for P450-mediated catalysis (Omura and Sato (1964)(See FIG. 18). This spectral assay indicates a clear absence ofabsorbance at 420 nm and documents the fact that normally labileproteins, such as P450s, are incorporated in their native form intoNanodiscs suitable for subsequent fractionation and biochemicalanalysis. To further demonstrate that the solubilized membrane proteinis accessible for binding substrate and suitable for use inhigh-throughput optical analysis, binding of xanthotoxin, one of severalfuranocoumarin substrates metabolized by this P450, to MSP1- andCYP6B1-containing Nanodiscs was analyzed in 96-well microtiter platesusing a sample volume of only 200 μl Nanodiscs (10 picomoles enzyme) andvarying concentrations of substrate. The Type-I binding spectra(Estabrook and Werringloer, 1978) obtained at varying concentrations ofxanthotoxin show an absorbance shift from 420 nm to 390 nm that ischaracteristic of substrates effectively displacing water as the sixthligand to the heme iron in the P450 catalytic site and converting theiron from low spin to high spin. The data presented in FIG. 18 clearlyillustrate that the ability of CYP6B1 to bind substrate is maintainedthroughout the Nanodisc assembly and subsequent fractionation process.

In summary, the present invention provides an important tool for thestudy of membrane protein targets as well as the complicatedmulti-component assemblies present in cellular bilayers. When coupledwith our ability to express individual cloned P450s or other membraneproteins in the frequently used baculovirus, yeast and mammalianexpression systems, these technologies present the opportunity todisplay single membrane proteins supported in native membrane bilayersin the development of biochemical methodologies previously restricted tosoluble proteins. The lipid composition of the particles derived fromMSP and membranes or membrane fragments mimics that of the startingmembranes or fragments, especially where solubilized membrane ormembrane fragment preparations are used as the source of thephospholipid(s) and hydrophobic protein or other hydrophobic molecule ofinterest. This contributes to maintaining the native conformation andactivity of the membrane (or other hydrophobic) protein which becomesincorporated into the particles with MSP.

The ability to bind substrates, inhibitors and other interactingmolecules with these solubilized membrane proteins using sensitiveoptical difference spectra in microtiter plates enables the developmentof high throughput screening methods for many different types ofmembrane proteins. For instance, cytochrome P450 and its reductasestabilized in a functional state through incorporation into Nanodiscsoffer an attractive means for measurements of drug metabolism andpharmokinetics, with applications in the pharmaceutical industry. Thefact that the Nanodisc solubilization procedures can be appliednonspecifically to all membrane proteins means that this technology canbe used to solubilize and fractionate many pharmacological targetproteins directly out of cellular membranes. Coupled with the histidine(or other) tag on the MSP molecule, this technology enables theimmobilization of target proteins on surfaces suitable for highthroughput screening. All the MSPs described herein can be used inpreparing Nanodiscs with purified and solubilized hydrophobic orpartially hydrophobic proteins or with hydrophobic or partiallyhydrophobic membrane proteins solubilized from membrane or membranefragment preparations.

Immunogenic Compositions

Antigens which are hydrophobic or partially hydrophobic can beformulated into immunogenic compositions for administration to a humanor animal in which an immune response, either cellular or humoral, isdesired. The incorporation of the antigen into a Nanodisc with a MSP ofthe present invention allows the preparation of stable aqueouspreparations which do not have a tendency to aggregate. At least oneantigenic determinant of the antigen is presented to the aqueous phase,with the more hydrophobic portions of the antigen being buried withinthe hydrophobic central region of the Nanodisc. The antigen incorporatedwithin the Nanodisc can be a protein, such as a cell membrane protein ora viral envelope protein, or it can be a lipopolysaccharide or alipooligosaccharide.

The antigen can be derived from a virus, especially an enveloped virus,a bacterium including, but not limited to, a bacterium, fungus,protozoan, parasite, or it can be derived from a particular type oftumor or cancer. The antigen-containing Nanodisc preparation can beadministered in prophylactic or therapeutic treatment regimens togenerate an immune response, and administration of these Nanodiscs canbe carried out in combination with other vaccine preparations forpriming and/or boosting.

Cancers (neoplastic conditions) from which cells can be obtained for useas an antigen source in the methods of the present invention includecarcinomas, sarcomas, leukemias and cancers derived from cells of thenervous system. These include, but are not limited to bone cancers(osteosarcoma), brain cancers, pancreatic cancers, lung cancers such assmall and large cell adenocarcinomas, rhabdosarcoma, mesiothelioma,squamous cell carcinoma, basal cell carcinoma, malignant melanoma, otherskin cancers, bronchoalveolar carcinoma, colon cancers, othergastrointestinal cancers, renal cancers, liver cancers, breast cancers,cancers of the uterus, ovaries or cervix, prostate cancers, lymphomas,myelomas, bladder cancers, cancers of the reticuloendothelial system(RES) such as B or T cell lymphomas, melanoma, and soft tissue cancers.

The terms “neoplastic cell”, “tumor cell”, or “cancer cell”, used eitherin the singular or plural form, refer to cells that have undergone amalignant transformation that makes them harmful to the host organism.Primary cancer cells (that is, cells obtained from near the site ofmalignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e.g., by such procedures as CAT scan, magneticresonance imaging (MRI), X-ray, ultrasound, or palpation. Biochemical orimmunologic findings alone may be insufficient to meet this definition.

Pathogens to which multiple antigen immunological responses areadvantageous include viral, bacterial, fungal and protozoan pathogens.Viruses to which immunity is desirable include, but are not limited to,hemorrhagic fever viruses (such as Ebola virus), immune deficiencyviruses (such as feline or human immunodeficiency viruses),herpesviruses, coronaviruses, adenoviruses, poxviruses, picornaviruses,orthomyxoviruses, paramyxoviruses, rubella, togaviruses, flaviviruses,bunyaviruses, reoviruses, oncogenic viruses such as retroviruses,pathogenic alphaviruses (such as Semliki forest virus or Sindbis virus),rhinoviruses, hepatitis viruses (Group B, C, etc), influenza viruses,among others. Bacterial pathogens to which immune responses are helpfulinclude, without limitation, staphylococci, streptococci, pneumococci,salmonellae, escherichiae, yersiniae, enterococci, clostridia,corynebacteria, hemophilus, neisseriae, bacteroides, francisella,legionella, pasteurellae, brucellae, mycobacteriae, bordetella,spirochetes, actinomycetes, chlamydiae, mycoplasmas, rickettsias, andothers. Pathogenic fungi of interest include but are not limited toCandida, cryptococci, blastomyces, histoplasma, coccidioides,phycomycetes, trichodermas, aspergilli, pneumocystis, and others.Protozoans to which immunity is useful include, without limitation,toxoplasma, plasmodia, schistosomes, amoebae, giardia, babesia,leishmania, and others. Other parasites include the roundworms,hookworms and tapeworms, filiaria and others.

A further object of the present invention is the administration of theantigen-containing immunogenic Nanodisc compositions of the presentinvention to a human or animal (e.g. horse, pig, cow, goat, rabbit,mouse, hamster) to generate immune responses, such as production ofantibody specific to the antigen or a cellular response such that cellsor tissues sharing the antigen are the subject of a cellular orcytotoxic immune response. Sera or cells collected from such humans oranimals are useful in providing polyclonal sera or cells for theproduction of hybridomas that generate monoclonal sera, such antibodypreparations being useful in research, diagnostic, and therapeuticapplications.

While the generation of an immune response includes at least some levelof protective immunity directed to the tumor cell (or neoplasticcondition), pathogen or parasite, the clinical outcome in the patientsuffering from such a neoplastic condition or infection with a parasiteor a pathogen can be improved by also treating the patient with asuitable chemotherapeutic agent, as known to the art. Where the pathogenis viral, an anti-viral compound such as acyclovir can be administeredconcomitantly with antigen-containing Nanodisc vaccination in patientswith herpes virus infection, or HAART (highly active anti-retroviraltherapy) in individuals infected with HIV. Where the pathogen is abacterial pathogen, an antibiotic to which that bacterium is susceptibleis desirably administered and where the pathogen is a fungus, a suitableantifungal antibiotic is desirably administered.

Similarly, chemical agents for the control and/or eradication ofparasitic infections are known and are advantageously administered tothe human or animal patients using dosages and schedules well known tothe art. Where the patient is suffering from a neoplastic condition, forexample, a cancer, the administration of the immunogenic compositioncomprising the Nanodiscs carrying one or more multiplicity ofcancer-associated antigens in the patient to which it has beenadministered is desirably accompanied by administration ofantineoplastic agent(s), including, but not limited to, suchchemotherapeutic agents as daunorubicin, taxol, thioureas,cancer-specific antibodies linked with therapeutic radionuclides, withthe proviso that the agent(s) do not ablate the ability of the patientto generate an immune response to the administered Nanodiscs and theantigens whose expression they direct in the patient. Nucleic acids formodulating gene expression or for directing expression of a functionalprotein can be incorporated within Nanodiscs, especially where thenucleic acid molecules are complexed with a cationic lipids, many ofwhich are commercially available.

Pharmaceutical formulations, such as vaccines or other immunogeniccompositions, of the present invention comprise an immunogenic amount ofthe antigen-bearing Nanodiscs in combination with a pharmaceuticallyacceptable carrier. An “immunogenic amount” is an amount of theantigen-bearing Nanodiscs which is sufficient to evoke an immuneresponse in the subject to which the pharmaceutical formulation isadministered. An amount of from about 10³ to about 10¹¹ particles perdose, preferably 10⁵ to 10⁹, is believed suitable, depending upon theage and species of the subject being treated. Depending on the settingfor administration (i.e., disease treatment or prevention), the dose(and repetition of administration) can be chosen to be therapeuticallyeffective or prophylactically effective.

Exemplary pharmaceutically acceptable carriers include, but are notlimited to, sterile pyrogen-free water and sterile pyrogen-freephysiological saline solution. Subjects which may be administeredimmunogenic amounts of the antigen-carrying Nanodiscs of the presentinvention include, but are not limited to, human and animal (e.g., dog,cat, horse, pig, cow, goat, rabbit, donkey, mouse, hamster, monkey)subjects. Immunologically active compounds such as cytokines and/or BCGcan also be added to increase the immune response to the administeredimmunogenic preparation.

Immunogenic compositions comprising the Nanodiscs which incorporateantigens of interest produced using the methods of the present inventionmay be formulated by any of the means known in the art. Suchcompositions, especially vaccines, are typically prepared asinjectables, either as liquid solutions or suspensions. Solid formssuitable for solution in, or suspension in, liquid prior to injectionmay also be prepared.

The active immunogenic ingredients (the Nanodiscs) are advantageouslymixed with excipients or carriers that are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients includebut are not limited to sterile water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof.

In addition, if desired, the immunogenic compositions, includingvaccines, may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, and/or adjuvantswhich enhance the effectiveness of the vaccine. Examples of adjuvantswhich may be effective include but are not limited to aluminumhydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE); and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against the immunogeniccomponent of the nanoscale particles after administration. Suchadditional formulations and modes of administration as known in the artmay also be used.

The immunogenic (or otherwise biologically active) antigen-containingNanodisc compositions are administered in a manner compatible with thedosage formulation, and in such amount as will be prophylacticallyand/or therapeutically effective. The quantity to be administered, whichis generally in the range of about 10³ to about 10¹⁰ particles,preferably 10⁵ to 10⁸, in a dose, depends on the subject to be treated,the capacity of the individual's immune system to synthesize antibodies,and the degree of protection desired. Precise amounts of the activeingredient required to be administered may depend on the judgment of thephysician, veterinarian or other health practitioner and may be peculiarto each individual, but such a determination is within the skill of sucha practitioner.

The vaccine or other immunogenic composition may be given in a singledose or multiple dose schedule. A multiple dose schedule is one in whicha primary course of vaccination may include 1 to 10 or more separatedoses, followed by other doses administered at subsequent time intervalsas required to maintain and or reinforce the immune response, e.g., atweekly, monthly or 1 to 4 months for a second dose, and if needed, asubsequent dose(s) after several months or years. Hydrophobic orpartially hydrophobic antigens can be incorporated into Nanodiscs asdescribed for other molecules (such as membrane proteins or smallmolecules). Where the antigen is in nature associated with or is withina membrane, either a solubilized pure or partially pure preparation or asolubilized membrane or membrane fragment preparation can be used as thesource of the input antigen in the Nanodisc assembly mixture.

Nuclear Magnetic Resonance

A current method for the diagnosis of myocardial ischemia utilizes anNMR relaxation agent containing gadolinium (Gd). The current marketleader is Magnevist (Trademark of Berlex). The Gd metal is chelated inthe form of gadopentetate dimeglumine. Unfortunately, the half life ofthis compound is only a few minutes in humans, due to its small size andrapid clearance. Nanodiscs are believed to have a half life of severalhours in human plasma.

Various organic and inorganic complexes can be incorporated into theNanodisc bilayer by conjugation (covalent attachment) with fatty-acidlike chains that then partition into the Nanodisc bilayer. We have usedthis technique to affix fluorescent molecules to the Nanodisc at variousloadings. For a typical 10 nm diameter Nanodisc containing about 160DPPC phospholipid molecules, up to about 40-50 such alkyl chain-anchoredspecies can be incorporated, replacing the native phospholipids, withoutcomprising the Nanodisc structure. This same procedure can be used toaffix other organics or inorganics to the Nanodisc, wherein the Nanodiscthen becomes a carrier of the compound and conveys the advantageouslycontrolled circulation lifetime while providing small and robust size.Such compounds include sugars, imaging agents, lipophilic dyes,photoactive (photodynamic) agents, etc. Photodynamic agents include, butare not limited to, those useful for treating tumors or atheroscleroticplaques, for example, porphyrins and phthalacyanin-related molecules.

Various chelating agents can be so constructed to provide a Nanodiscwith approximately 50 Gd relaxation agents in a 10 nm diameter package.This should have great benefit in providing a longer lifetime imagingagent for cardiovascular imaging. We have completed a first experimentalong these lines using a commercially available chelating agent, butwhich provides an incomplete coordination of the Gd molecule. Thiscompound then is prone to precipitation. It is straightforward chemistry(J. Med. Chem 42, 2852 (1999)) to affix a long alkyl chain to theMagnevist structure, for example at the methylene carbon position, tohave the same chelating properties as Magnevist but now in a moreconcentrated entity with increased plasma lifetime.

Functional Equivalents

It is understood that a variant of a specifically exemplified MSP can bemade with an amino acid sequence which is substantially identical (atleast about 80 to 99% identical, and all integers therebetween) to theamino acid sequence to an MSP of the present invention and it forms afunctionally equivalent, amphiphilic, three dimensional structure andretains the ability to form Nanodiscs with phospholipid and/or apassenger molecule such as a hydrophobic or partially hydrophobicprotein, among others. It is well known in the biological arts thatcertain amino acid substitutions can be made in protein sequenceswithout affecting the function of the protein.

Generally, conservative amino acid substitutions or substitutions ofsimilar amino acids are tolerated without affecting protein function.Similar amino acids can be those that are similar in size and/or chargeproperties, for example, aspartate and glutamate and isoleucine andvaline are both pairs of similar amino acids. Nonpolar amino acidsinclude alanine, valine, leucine, phenylalanine, tryptophan, methionine,isoleucine, cysteine and glycine. Uncharged polar amino acids includeserine, threonine, asparagine, glutamine and tyrosine. Charged polarbasic amino acids include lysine, arginine and histidine. Substitutionsof one for another are permitted when helix formation is not disruptedexcept as intended. Similarity between amino acid pairs has beenassessed in the art in a number of ways. For example, Dayhoff et al.(1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement3, Chapter 22, pages 345-352, which is incorporated by reference herein,provides frequency tables for amino acid substitutions which can beemployed as a measure of amino acid similarity. Dayhoff et al.'sfrequency tables are based on comparisons of amino acid sequences forproteins having the same function from a variety of evolutionarilydifferent sources.

Substitution mutation, insertional, and deletional variants of thedisclosed nucleotide (and amino acid) sequences can be readily preparedby methods which are well known to the art. These variants can be usedin the same manner as the exemplified MSP sequences so long as thevariants have substantial sequence identity with a specificallyexemplified sequence of the present invention. As used herein,substantial sequence identity refers to homology (or identity) which issufficient to enable the variant polynucleotide or protein to functionin the same capacity as the polynucleotide or protein from which thevariant is derived. Preferably, this sequence identity is greater than70% or 80%, more preferably, this identity is greater than 85%, or thisidentity is greater than 90%, and or alternatively, this is greater than95%, and all integers between 70 and 100%. It is well within the skillof a person trained in this art to make substitution mutation,insertional, and deletional mutations which are equivalent in functionor are designed to improve the function of the sequence or otherwiseprovide a methodological advantage. No variants which may read on anynaturally occurring proteins or which read on a prior art variant areintended to be within the scope of the present invention as claimed.

It is well known in the art that the polynucleotide sequences of thepresent invention can be truncated and/or mutated such that certain ofthe resulting fragments and/or mutants of the original full-lengthsequence can retain the desired characteristics of the full-lengthsequence. A wide variety of restriction enzymes which are suitable forgenerating fragments from larger nucleic acid molecules are well known.In addition, it is well known that Ba/31 exonuclease can be convenientlyused for time-controlled limited digestion of DNA. See, for example,Maniatis (1982) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, pages 135-139, incorporated herein byreference. See also Wei et al. (1983 J. Biol. Chem. 258:13006-13512. Byuse of Ba/31 exonuclease (commonly referred to as “erase-a-base”procedures), the ordinarily skilled artisan can remove nucleotides fromeither or both ends of the subject nucleic acids to generate a widespectrum of fragments which are functionally equivalent to the subjectnucleotide sequences. One of ordinary skill in the art can, in thismanner, generate hundreds of fragments of controlled, varying lengthsfrom locations all along the original MSP-encoding sequence. Theordinarily skilled artisan can routinely test or screen the generatedfragments for their characteristics and determine the utility of thefragments as taught herein. It is also well known that the mutantsequences of the full length sequence, or fragments thereof, can beeasily produced with site directed mutagenesis. See, for example,Larionov, O. A. and Nikiforov, V. G. (1982) Genetika 18(3):349-59;Shortle, D, DiMaio, D., and Nathans, D. (1981) Annu. Rev. Genet.15:265-94; both incorporated herein by reference. The skilled artisancan routinely produce deletion-, insertion-, or substitution-typemutations and identify those resulting mutants which contain the desiredcharacteristics of the full length wild-type sequence, or fragmentsthereof, i.e., those which retain GIcNAc T-Vb activity.

DNA sequences having at least 70, 80, 85, 90 or 95% or greater identityto the recited DNA coding sequence of Tables 1, 3, 4 or 5 (SEQ ID NOs:1,3, 7 or 9) and functioning to encode a GIcNAc T-Vb protein are withinthe scope of the present invention. Functional equivalents are includedin the definition of a GIcNAc T-Vb encoding sequence. Following theteachings herein and using knowledge and techniques well known in theart, the skilled worker will be able to make a large number of operativeembodiments having equivalent DNA sequences to those listed hereinwithout the expense of undue experimentation.

As used herein percent sequence identity of two nucleic acids isdetermined using the algorithm of Altschul et al. (1997) Nucl. AcidsRes. 25: 3389-3402; see also Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.(1990) J. Mol. Biol. 215:402-410. BLAST nucleotide searches areperformed with the NBLAST program, score=100, wordlength=12, to obtainnucleotide sequences with the desired percent sequence identity. Toobtain gapped alignments for comparison purposes, Gapped BLAST is usedas described in Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (NBLAST and XBLAST) are used. See theNational Center for Biotechnology Information on the internet.

Antibody Technology

Monoclonal or polyclonal antibodies, preferably monoclonal, specificallyreacting with an MSP of the present invention (or to another protein ofinterest) can be made by methods known in the art. See, e.g., Harlow andLane (1988) Antibodies: A Laboratory Manual, Cold Spring HarborLaboratories; Goding (1986) Monoclonal Antibodies: Principles andPractice, 2d ed., Academic Press, New York; and Ausubel et al. (1993)Current Protocols in Molecular Biology, Wiley Interscience, New York,N.Y.

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993)Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al.(eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.)Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oldand Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkeley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic AcidHybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York; and Ausubel et al. (1992) Current Protocols in MolecuiarBiology, Greene/Wiley, New York, N.Y. Abbreviations and nomenclature,where employed, are deemed standard in the field and commonly used inprofessional journals such as those cited herein.

All references cited in the present application are incorporated byreference herein to the extent that there is no inconsistency with thepresent disclosure.

The description provided herein is not intended to limit the scope ofthe invention as claimed herein. Any variations in the exemplifiedarticles and methods which occur to the skilled artisan are intended tofall within the scope of the present invention.

EXAMPLES Example 1 Construction of Recombinant DNA Molecules forExpression of MSPs

The human proapo A-I coding sequence as given below was inserted betweenNcol and Hindlul sites (underlined) in pET-28 (Novagen, Madison, Wis.).Start and stop codons are in bold type. The restriction endonucleaserecognition sites used in cloning are underlined.

TABLE 1 ProApo A-I coding sequence (SEQ ID NO:1) Restriction sites usedin cloning are underlined, and the translation start and stop signalsare shown in bold. CCATGGCCCATTTCTGGCAGCAAGATGAACCCCCCCAGAGCCCCTGGGATCGAGTGAAGGACCTGGCCACTGTGTACGTGGATGTGCTCAAAGACAGCGGCAGAGACTATGTGTCCCAGTTTGAAGGCTCCGCCTTGGGAAAACAGCTAAACCTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAAGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTAATAAGCTT-3′

TABLE 2 ProApo A-I amino acid sequence (SEQ ID NO:2)MAHFWQQDEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

The construction of the MSP1 coding sequence was accomplished asfollows. Primers were designed to produce DNA encoding MSP1, thetruncated protein lacking the N-terminal domain of proApo A-I, bypolymerase chain reaction (PCR) mutagenesis (Higuchi et al., 1988).

-   Primer 1 (SEQ ID NO:3)-   (5′-TATACCATGGGCCATCATCATCATCATCATATAGAAGGAA    GACTAAAGCTCCTTGACAACT-3′) introduces an N-terminal 6-histidine tag    for purification and manipulation of MSP1, and a factor Xa cleavage    site for removal of the histidine tag. Factor Xa cleaves after R in    the protein sequence IEGR.-   Primer 2 (SEQ ID NO:4) (5′-GCAAGCTTATTACTGGGTGTTGAGCTTCTT-3′) was    used as a reverse primer.

TABLE 3 Histidine-tagged MSP1 coding sequence (SEQ ID NO:5). Restrictionsites used in cloning are underlined, and the translation start and stopsignals are shown in bold.TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGTTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAACCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTAATAAGCTTGC

TABLE 4 Histidine-tagged MSP1 amino acid sequence (SEQ ID NO:6)MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

For production of MSP1 without a N-terminal histidine tag, primer 1 wasreplaced with primer 1a: 5′-TACCATGGCAAAGCTCCTTGACAACTG-3′ (SEQ ID NO:7)to produce the sequence provided in SEQ ID NO:8.

TABLE 5 Non-Histidine-tagged MSP1 DNA sequence (SEQ ID NO:8).Restriction sites used in cloning are underlined, and the translationstart and stop signals are shown in bold.TACCATGGCAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGTTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAACCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTAATAAGCTTGC

TABLE 6 Non-Histidine-tagged MSP1 amino acid sequence (SEQ ID NO:9).MAKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

The production of an MSP with tandem repeats (MSP2) was carried at asdescribed below. The following primers were used to generate MSP2 (seeFIGS. 6A-6B):

Primer 3 (SEQ ID NO:10): 5′-TACCATGGCAAAGCTCCTTGACAACTG-3′ primer3a (SEQID NO:11): 5′-TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCCTTGACAACT-3′ Primer 4 (SEQ ID NO:12):5′-TAAGAAGCTCAACACCCAGGGTACCGGTGGAGGTAGTGGAGGTGGTACCCTA-3′ Primer 5 (SEQID NO:13): 5′-CAGGGTACCGGTGGAGGTAGTGGAGGTGGTACCCTAAAGCTCCTTGACAA-3′Primer 6 (SEQ ID NO:14): 5′-GCAAGCTTATTACTGGGTGTTGAGCTTCTT-3′

In a first PCR, primer 2 (or primer 2a for N-terminal histidine tag) andprimer 4 were used to add a linker sequence (encoding the amino acidsequence GTGGGSGGGT; SEQ ID NO:15) to the 3′ end of the MSP gene toproduce MSP-A. In a second PCR, the linker was added to the 5′ end ofthe MSP gene to produce MSP-B. Treatment of MSP-A and MSP-B with KpnIand subsequent ligation produced the following constructs, one with andone without the linker. The Kpn I site provides an easy way to insertingany desired linker sequence by restriction with Kpn I and religationwith double-stranded synthetic DNA encoding desired linker. See FIGS.6A-6B.

TABLE 7 MSP2 (with histidine tag, without long linker) DNA sequence (SEQID NO:16). The translation start and stop codons are in bold type, andthe restriction endonuclease recognition sites used in cloning areunderlined. TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGGGTACCCTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCA GTAATAAGCTTGC

TABLE 8 MSP2 (with histidine tag, without long linker) amino acidsequence (SEQ ID NO:17)MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

TABLE 9 MSP2L(with histidine tag, with long linker) DNA sequence (SEQ IDNO:18). Translation start and stop codons are in bold type; restrictionendonuclease sites used in cloning are underlined.TACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGGGTACCGGTGGAGGTAGTGGAGGTGGTACCCTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTAATAAGCTTGC

TABLE 10 MSP2 (with histidine tag, with long linker, in bold type) aminoacid sequence (SEQ ID NO:19).MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTGGGSGGGTLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

To delete hinge regions, deletion of helices 4 and 5 was carried out byconstructing the C-terminal portion of MSP1 using the following PCRprimers and the Sac I and Hind III fragment of the MSP1 coding sequenceas template.

Primer A (SEQ ID NO:20):5′-TGGAGCTCTACCGCCAGAAGGTGGAGCCCTACAGCGACGAGCT-3′ Primer B (SEQ IDNO:21): 5′-GCAAGCTTATTACTGGGTGTTGAGCTTCTT-3′.

This amplification product was digested with Sacl and Hindlil andligated into pLitmus 28 for sequencing. The Sac I+HindIII treatedhistidine-tagged MSP1 construct in pET 28 vector was then ligated withthe above fragment to produce MSP1Da.

TABLE 11 MSP1D5D6 DNA sequence (SEQ ID NO:22). Translations start andstop codons are in bold type; restriction endonuclease recognition sitesare underlined. TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTctaccgccagaaggtggagcCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAACCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTAATAAGCTTGC

TABLE 12 MSP1D5D6 amino acid sequence (SEQ ID NO:23).MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

Deletion of helices 5 and 6 was performed in a similar manner, but twoseparate PCR steps using the following primers were employed in a firstreaction (Reaction 1,

Primer C: 5′-CAGAATTCGCTAGCCGAGTACCACGCCAA-3′, SEQ ID NO:24; and PrimerD: 5′-GCAAGCTTATTACTGGGTGTTGAGCTTCTT-3′, SEQ ID NO:25) and a secondreaction (Reaction 2, Primer E: 5′-ATACCATGGGCCATCATCATCATCATCATA-3′,SEQ ID NO:26; and Primer F: 5′-CAGAATTCGCTAGCCTGGCGCTCAACTTCTCTT-3′, SEQID NO:27.

The PCR products encode the N- and C-terminal portions of an MSP bothlacking helices 5 and 6 and each contain a Nhel restriction site. Afterdigestion of the PCR products with NheI, NcoI and HindIII, the fragmentswas ligated into Ncol+HindIII treated pET 28 to produce the DNA sequenceof MSP1D6D7 See FIGS. 9A-9B.

TABLE 13 MSP1D6D7 DNA sequence (SEQ ID NO:28). Translation start andstop codons are shown in bold type, and restriction endonucleaserecognition sites used in cloning are underlined.TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGTTGAGCGCCAGGCTAGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAACCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTAATAAGCTTGC

TABLE 14 MSP1D6D7 amino acid sequence (SEQ ID NO:29).MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

Example 2 Construction of Synthetic MSP Gene

A synthetic gene for MSP1 is made using the following overlappingsynthetic oligonucleotides which are filled in using PCR. The codonusage has been optimized for expression in E. coli, and restrictionsites have been introduced for further genetic manipulations of thegene.

Synthetic nucleotide taps1a (SEQ ID NO:30)TACCATGGGTCATCATCATCATCATCACATTGAGGGACGTCTGAAGCTGTTGGACAATTGGGACTCTGTTACGTCTA Synthetic nucleotide taps2a (SEQ ID NO:31)AGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGGAAATGTC CAAAGATSynthetic nucleotide taps3a (SEQ ID NO:32)TATCTAGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAA Syntheticnucleotide taps4a (SEQ ID NO:33)ATGAGCTCCAAGAGAAGCTCAGCCCATTAGGCGAAGAAATGCGCGATCGCGCCCGTGCACATGTTGATGCACT Synthetic nucleotide taps5a (SEQ ID NO:34)GTCTCGAGGCGCTGAAAGAAAACGGGGGTGCCCGCTTGGCTGAGTACCACGCGA AAGCGACAGAASynthetic nucleotide taps6a (SEQ ID NO:35)GAAGATCTACGCCAGGGCTTATTGCCTGTTCTTGAGAGCTTTAAAGTCAGTTTTCT Syntheticnucleotide taps1b (SEQ ID NO:36)CAGAATTCCTGCGTCACGGGGCCCAGTTGTTCGCGAAGTTTACTGAAGGTAGACG TAACAG Syntheticnucleotide taps2b (SEQ ID NO:37)TCATCTAGATATGGCTGAACCTTGGCCTTCACCTCTTCTAAATCTTTGGACATTT Syntheticnucleotide taps3b (SEQ ID NO:38)TGGAGCTCATGGAGTTTTTGGCGTGCCCCCTCTTGCAGTTCCGCACGCAGCGGTTCCACCTTTTGACGATATAATTCCAT Synthetic nucleotide taps4b (SEQ ID NO:39)GCCTCGAGACGTGCGGCCAAACGCTGGCGAAGTTCATCCGAATACGGCGCCAAATGAGTCCGGAGTGCATCAACAT Synthetic nucleotide taps5b (SEQ ID NO:40)GTAGATCTTCCAGCGCCGGTTTCGCTTTTTCGCTCAAGGTGCTCAGGTGTTCTGTC GCTTT Syntheticnucleotide taps6b (SEQ ID NQ:41)CCAAGCTTATTACTGGGTATTCAGCTTTTTAGTATATTCTTCCAGAGCTGACAGAAA ACTGACTTT

TABLE 15 Full synthetic gene sequence for MSP1 (SEQ ID NO:42).Restriction sites used in cloning are underlined, and the translationstart and stop signals are shown in bold.ACCATGGGTCATCATCATCATCATCACATTGAGGGACGTCTGAAGCTGTTGGACAATTGGGACTCTGTTACGTCTACCTTCAGTAAACTTCGCGAACAACTGGGCCCCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGGAAATGTCCAAAGATTTAGAAGAGGTGAAGGCCAAGGTTCAGCCATATCTAGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAAAAGGTGGAACCGCTGCGTGCGGAACTGCAAGAGGGGGCACGCCAAAAACTCCATGAGCTCCAAGAGAAGCTCAGCCCATTAGGCGAAGAAATGCGCGATCGCGCCCGTGCACATGTTGATGCACTCCGGACTCATTTGGCGCCGTATTCGGATGAACTTCGCCAGCGTTTGGCCGCACGTCTCGAGGCGCTGAAAGAAAACGGGGGTGCCCGCTTGGCTGAGTACCACGCGAAAGCGACAGAACACCTGAGCACCTTGAGCGAAAAAGCGAAACCGGCGCTGGAAGATCTACGCCAGGGCTTATTGCCTGTTCTTGAGAGCTTTAAAGTCAGTTTTCTGTCAGCTCTGGAAGAATATACTAAAAAGCTGAATACCCAGTAATAAGCTTGG

The following is the amino acid sequence of a MSP polypeptide in whichhalf repeats are deleted:

TABLE 16 MSP1D3 (SEQ ID NO:43).MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

TABLE 17 MSP1D9 (SEQ ID NO:44).MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPVLESFKVSFLSALEEYTKKLNTQ

TABLE 18 MSP tandem repeat with first half-repeats deleted (MSP2delta1)(SEQ ID NO:45) MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

Plasmids for the expression of extended MSPs were constructed fromplasmid for MSP1 described in Bayburt et al. (2002) Nanoletters2:853-856 using a “Seamless” cloning kit (Stratagene) according to themanufacturer recommendations. An alternative N-terminus for MSP1TEV wasadded by PCR; the primers were designed to include Nco I and Hind IIIrestriction sites. The PCR product was cloned into the pET28a plasmid(Novagen). Truncated mutants of MSP were produced with a Quick-changekit (Stratagene) using the MSP1TEV plasmid as a template. The presenceof the desired insertions or deletions and absence of PCR-inducedmutations were verified by DNA sequencing.

Expression and purification of the MSP proteins was performed asdescribed herein. Protein purity was characterized by SDS-PAGE andElectrospray Mass Spectrometry; it was found to be greater than 95%. TheTEV protease expression system was purchased (Science Reagents, Inc.,Atlanta, Ga.) and used after some minor modifications. The sequences ofnew scaffold proteins were optimized with respect to salt link scoresfor the belt model of the antiparallel dimer as described in Segrest etal. (1999) J. Biol. Chem. 274:31755-31758. At first, the amino acidsequences of the extended mutants were generated so that each of thecentral helices (from H3 to H7) (see FIG. 19), was inserted sequentiallyat every position between other central helices, i.e. after H3, H4, H5,and H6, and the number of favorable salt links minus number ofunfavorable contacts of the same charges was calculated for all possibleconfigurations of antiparallel dimers in the resulting scaffold protein(Segrest (1999) supra). As a result, the insertion mutants shown at FIG.20 were selected as optimal for maximum salt link scores. These extendedscaffold proteins, as well as truncated scaffold proteins, alsocontaining different tag sequences at the N. terminus, were engineeredin E. coli and expressed with a high yield and purified by standardprocedures.

With reference to the following protein and DNA sequences, the MSPs wehave utilized can be summarized as the following linked structures. NoteH1, H2 refer to the sequences of Helix #1 etc. His is a (His)6 tag, TEVis the tobacco viral protease, X is the Factor X (ten) protease site.

TABLE 19 Amino Acid Sequences of MSP Building Blocks GLOBDEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLN (SEQ ID NO:89) HisXMGHHHHHHIEGR (SEQ ID NO:47) HisTEV MGHHHHHHHDYDIPTTENLYFQG (SEQ IDNO:48) Helix 1 (H1): LKLLDNWDSVTSTFSKLREQLG (SEQ ID NO:49) Helix 2 (H2):PVTQEFWDNLEKETEGLRQEMS (SEQ ID NO:50) Helix 3 (H3): KDLEEVKAKVQ (SEQ IDNO:51) Helix 4 (H4): PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO:52) Helix 5 (H5):PLRAELQEGARQKLHELQEKLS (SEQ ID NO:53) Helix 6 (H6):PLGEEMRDRARAHVDALRTHLA (SEQ ID NO:54) Helix 7 (H7):PYSDELRQRLAARLEALKENGG (SEQ ID NO:55) Helix 8 (H8):ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO:56) Helix 9 (H9): PALEDLRQGLL (SEQ IDNO:57) Helix 10 (H10): PVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO:58) Helix 0.5(H0.5): STFSKLREQLG (SEQ ID NO:59) Helix 10.5 (H10.5): SALEEYTKKLNTQ(SEQ ID NO:87) Helix 2S (H2): PVTQEFWDNLEKETEGLRQEMS (SEQ ID NO:136)

TABLE 20 Sequences encoding the MSP Building Blocks of TABLE 19. HisXATGGGTCATCATCATCATCATCACATTGAG (SEQ ID NO:60) GGACGT HisTEVATGGGTCATCATCATCATCATCATCACGA (SEQ ID NO:61)TTATGATATTCCTACTACTGAGAATTTGT ATTTTCAGGGT Helix 1 (H1):CTGAAGCTGTTGGACAATTGGGACTCTGT (SEQ ID NO:62)TACGTCTACCTTCAGTAAACTTCGCGAAC AACTGGGC Helix 2 (H2):CCCGTGACGCAGGAATTCTGGGACAACCT (SEQ ID NO:63)GGAAAAAGAAACCGAGGGACTGCGTCAGG AAATGTCC Helix 3 (H3):AAAGATTTAGAAGAGGTGAAGGCCAAGGT (SEQ ID NO:64) TCAG Helix 4 (H4):CCATATCTCGATGACTTTCAGAAAAAATG (SEQ ID NO:65)GCAGGAAGAGATGGAATTATATCGTCAAA AGGTGGAA Helix 5 (H5):CCGCTGCGTGCGGAACTGCAAGAGGGGGC (SEQ ID NO:66)ACGCCAAAAACTCCATGAGCTCCAAGAGA AGCTCAGC Helix 6 (H6):CCATTAGGCGAAGAAATGCGCGATCGCGCC (SEQ ID NO:67)CGTGCACATGTTGATGCACTCCGGACTCA TTTGGCG Helix 7 (H7):CCGTATTCGGATGAACTTCGCCAGCGTTTG (SEQ ID NO:68)GCCGCACGTCTCGAGGCGCTGAAAGAAAAC GGGGGT Helix 8 (H8):GCCCGCTTGGCTGAGTACCACGCGAAAGC (SEQ ID NO:69)GACAGAACACCTGAGCACCTTGAGCGAAA AAGCGAAA Helix 9 (H9):CCGGCGCTGGAAGATCTACGCCAGGGCTT (SEQ ID NO:70) ATTG Helix 10 (H10):CCTGTTCTTGAGAGCTTTAAAGTCAGTTT (SEQ ID NO:71)TCTGTCAGCTCTGGAAGAATATACTAAAA AGCTGAATACCCAG Helix 0.5 (H0.5):TCTACCTTCAGTAAACTTCGCGAACAACT (SEQ ID NO:72) GGGC Helix 10.5 (H10.5):CAGTTTTCTGTCAGCTCTGGAAGAATATA (SEQ ID NO:88) CTAAAAAGCTGAATACCCAG Helix2S (H2S): TCCGTGACGCAGGAATTCTGGGACAACCT (SEQ ID NO:90)GGAAAAAGAAACCGAGGGACTGCGTCAGG AAATGTCC

Several particular MSP sequences useful in the present invention are thefollowing combinations of the above sequences, as given in Table 21 andothers.

TABLE 21 Engineered MSPs Useful in Nanodisc Preparation. MSP1HisX-H1-H2-H3-H4-H5-H6-H7-H8-H9- (SEQ ID NO:6) H10 MSP1E1HisX-H1-H2-H3-H4-H4-H5-H6-H7-H8- (SEQ ID NO:73) H9-H10 MSP1E2HisX-H1-H2-H3-H4-H5-H4-H5-H6-H7- (SEQ ID NO:74) H8-H9-H10 MSP1E3HisX-H1-H2-H3-H4-H5-H6-H4-H5-H6- (SEQ ID NO:75) H7-H8-H9-H10 MSP1TEVHisTev-H1-H2-H3-H4-H5-H6-H7-H8-H9- (SEQ ID NO:76) H10 MSP1NHH1-H2-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO:77) MSP1T2HisTev-H0.5-H2-H3-H4-H5-H6-H7-H8- (SEQ ID NO:78) H9-H10 MSP1T2NHH0.5-H2-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO:79) MSP1T3HisTev-H2-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO:80) MSP1D3HisX-H1-H2-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO:43) MSP1D9HisX-H1-H2-H3-H4-H5-H6-H7-H8-H10 (SEQ ID NO:44) MSP1D5D6HisX-H1-H2-H3-H4-H7-H8-H9-H10 (SEQ ID NO:23) MSP1D6D7HisX-H1-H2-H3-H4-H5-H8-H9-H10 (SEQ ID NO:82) MSP1D3D9HisX-H1-H2-H4-H5-H6-H7-H8-H10 (SEQ ID NO:83) MSP1D10.5HisX-H1-H2-H3-H4-H5-H6-H7-H8-H9- (SEQ ID NO:84) H10.5 MSP1D3D10.5HisX-H1-H2-H4-H5-H6-H7-H8-H9-H10.5 (SEQ ID NO:85) MSP1T4HisTEV-H2S-H3-H4-H5-H6-H7-H8-H9- (SEQ ID NO:91) H10 Apo A-IGLOB-H1-H2-H3-H4-H4-H5-H6-H5-H6- H7-H8-H9-H10 MSP1T5HisTev-H2.5-H3-H4-H5-H6-H7-H8-H9- (SEQ ID NO:92) H10 MSP1T6HisTev-H3-H4-H5-H6-H7-H8-H9-H10 (SEQ ID NO:93) MSP1E3TEV:HisTev-H1-H2-H3-H4-H5-H6-H4-H5-H6- (SEQ ID NO:94) H7-H8-H9-H10 MSP1E3D1:HisTev-H0.5-H2-H3-H4-H5-H6-H4-H5- (SEQ ID NO:95) H6-H7-H8-H9-H10MSP2TEV: HisTev-H1-H2-H3-H4-H5-H6-H7-H8-H9- (SEQ ID NO:96)H10-GT-H1-H2-H3-H4-H5-H6-H7-H8-H9- H10 MSP1N1:His-TEV-H2S-H3-H4-H4-H5-H6-H7-H8- (SEQ ID NO:97) H9 MSP2N1:HisTev-H0.5-H2-H3-H4-H5-H6-H7-H8- (SEQ ID NO:98)H9-H10-GT-H0.5-H2-H3-H4-H5-H6-H7- H8-H9-H10 MSP2N2:HisTev-H0.5-H2-H3-H4-H5-H6-H7-H8- (SEQ ID NO:99)H9-H10-GT-H2-H3-H4-H5-H6-H7-H8- H9-H10

In addition to these sequences, there are two fusion protein (tandemrepeat MSP) constructs of reference. These are composed of two MSP1constructs linked by a Gly-Thr linker:

MSP2 (MSP1-Gly-Thr-MSP1, SEQ ID NO:17) and MSP2D1D1 (MSP1T3-Gly-Thr-H2-H3-H4-H5-H6-H7-H8-H9-H10, SEQ ID NO:86).

Other constructs that can be readily produced include permutations ofthe above, i.e., MSP1 or a tandemly repeated MSP with either a short orlong linker sequence with any combination of the following: hingedeletion, hinge replacement, half-repeat deletion, histidine tag,different linkers for MSP2 analogs.

The coding and amino acid sequences of MSP1T4 are given in Tables 22 and23, respectively.

TABLE 22 DNA sequence encoding MSP1T4 (SEQ ID NO:100)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag

TABLE 23 Amino acid sequence of MSP1T4 (SEQ ID NO:91)MGHHHHHHHDYDIPTTENLYFQGSVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQIn the schematic for MSP1T5, H2.5 indicates the second half of the H2helical sequence, i.e. the last 33 nucleotides or 11 amino acids is notincluded in the MSP sequence. The coding and amino acid sequence forthis protein is given in Tables 24 and 25, respectively.

TABLE 24 DNA sequence encoding MSP1T5 (SEQ ID NO:101)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggtaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag

TABLE 25 Amino acid sequence of MSP1T5 (SEQ ID NO:92)MGHHHHHHHDYDIPTTENLYFQGKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

TABLE 26 DNA sequence encoding MSP1T6 (SEQ ID NO:102)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggtaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag

TABLE 27 Amino acid sequence of MSP1T6 (SEQ ID NO:93)MGHHHHHHHDYDIPTTENLYFQGKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEY TKKLNTQ

MSP1T5 and MSP1T6 discs preps are not homogeneous under all assemblyconditions. The results are highly dependent on the particular assemblyconditions.

In the following MSP construct (MSP1N1), H10 is not included, and two H4motifs are inserted. The coding and amino acid sequences are given inTables 28 and 29, respectively. This MSP is designed to increase thenumber of possible salt bridges on the interhelical interface.

TABLE 28 DNA sequence encoding MSP1N1 (SEQ ID NO:103)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattg

TABLE 29 Amino acid sequence of MSP1N1 (SEQ ID NO:97)MGHHHHHHHDYDIPTTENLYFQGSVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLThe following “extended” MSPs incorporate a cleavable His-tag and use aTEV protease recognition site.

TABLE 30 DNA sequence encoding MSP1E3TEV(HisTev-H1-H2-H3-H4-H5-H6-H4-H5-H6-H7-H8-H9-H10) (SEQ ID NO:105)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggtctgaagctgttggacaattgggactctgttacgtctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag

TABLE 31 Amino acid sequence of MSP1E3TEV (SEQ ID NO:94)MGHHHHHHHDYDIPTTENLYFQGLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKL NTQ

TABLE 32 DNA sequence encoding MSP1E3D1 (SEQ ID NO:106)(HisTev-H0.5-H2-H3-H4-H5-H6-H4-H5-H6-H7-H8-H9-H10)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag

TABLE 33 Amino acid sequence of MSP1E3D1 (SEQ ID NO:95)MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQA protein corresponding to, MSP2 with a N-terminal TEV cleavable His-taghas been designed. The coding and amino acid sequences are given inTables 34 and 35, respectively.

TABLE 34 DNA sequence encoding MSP2TEV(HisTev-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10) (SEQ ID NO:107)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggtctaaagctccttgacaactgggacagcgtgacctccaccttcagcaagctgcgcgaacagctcggccctgtgacccaggagttctgggataacctggaaaaggagacagagggcctgaggcaggagatgagcaaggatctggaggaggtgaaggccaaggtgcagccctacctggacgacttccagaagaagtggcaggaggagatggagctctaccgccagaaggtggagccgctgcgcgcagagctccaagagggcgcgcgccagaagctgcacgagctgcaagagaagctgagcccactgggcgaggagalgcgcgaccgcgcgcgcgcccatgtggacgcgctgcgcacgcatctggccccctacagcgacgagctgcgccagcgcttggccgcgcgccttgaggctctcaaggagaacggcggcgccagactggccgagtaccacgccaaggccaccgagcatctgagcacgctcagcgagaaggccaagcccgcgctcgaggacctccgccaaggcctgctgcccgtgctggagagcttcaaggtcagcttcctgagcgctctcgaggagtacactaagaagctcaacacccagggtaccctaaagctccttgacaactgggacagcgtgacctccaccttcagcaagctgcgcgaacagctcggccctgtgacccaggagttctgggataacctggaaaaggagacagagggcctgaggcaggagatgagcaaggatctggaggaggtgaaggccaaggtgcagccctacctggacgacttccagaagaagtggcaggaggagatggagctctaccgccagaaggtggagccgctgcgcgcagagctccaagagggcgcgcgccagaagctgcacgagctgcaagagaagctgagcccactgggcgaggagatgcgcgaccgcgcgcgcgcccatgtggacgcgctgcgcacgcatctggccccctacagcgacgagctgcgccagcgcttggccgcgcgccttgaggctctcaaggagaacggcggcgccagactggccgagtaccacgccaaggccaccgagcatctgagcacgctcagcgagaaggccaagcccgcgctcgaggacctccgccaaggcctgctgcccgtgctggagagcttcaaggtcagcttcctgagcgctctcgaggagtacactaagaagctcaacacccag

TABLE 35 Amino acid sequence of HisTEV-MSP2 (SEQ ID NO:96)MGHHHHHHHDYDIPTTENLYFQGLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEYTKKLNTQGTLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

New constructs have been designed to produce a which make a “lineardimer” to generate Nanodiscs with only a single polypeptide sequence.These are fusions that make use of our knowledge of the parts of theMSP1 sequences which are important and are thus are “MSP2 derivatives”.All have the TEV protease-cleavage His-tag.

TABLE 36 DNA sequence encoding MSP2N1(HisTev-H0.5-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1/2-H2-H3-H4-H5-H6-H7-H8-H9-H10) (SEQ ID NO:108)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccagggtaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag

TABLE 37 Amino acid sequence of MSP2N1 (SEQ ID NO:98)MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

TABLE 38 DNA sequence encoding MSP2N2 (SEQ ID NO:109)(HisTev-H0.5-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H2-H3-H4-H5-H6-H7-H8-H9-H10)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccagggtacccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag

TABLE 39 Amino acid sequence of MSP2N2 (SEQ ID NO:99)MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

A further MSP2 derivative (MSP2N3 has been designed to include helices2-10 following the linker part of the H1 helix sequence. The DNA codingand amino acid sequences are given in Tables 40 and 41, respectively.

TABLE 40 DNA sequence encoding MSP2N3(HisTev-H0.5-H2-H3-H4-H5-H6-H7-H8-H9-H10-GTREQLG-H2-H3-H4-H5-H6-H7-H8-H9-H10) (SEQ ID NO:110)Atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccagggtacccgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccagtaagctt

TABLE 41 Amino acid sequence of MSP2N3 (SEQ ID NO:111)MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

Unlike MSP2 and MSP2TEV these proteins self-assemble with lipids at300:1 to 400:1 molar ratios with preferable formation of significantlybigger particles (Stokes diameter approximately 15.5 nm, correspondingto a calculated diameter assuming discoidal shape of about 17 nm).

New dimer sequences (i.e., tandem repeat MSP) have been designed withthe fusion region to be composed of two different linkers which havehigh propensity to form beta-turns (Creighton, Proteins, p. 226). Thesescaffold proteins are specifically designed to promote the anti-parallelhelix-turn-helix structure in Nanodiscs. The constituent scaffoldproteins include MSP1T3, as well as the specially designed new scaffoldproteins as described herein, MSP1N1 and the circularly permuted MSP2N5which has a modified sequence of amphipathic helices to optimize thesalt bridges formed between two scaffold proteins in the antiparallelhelix-turn-helix structure.

The general scheme for a tandem repeat MSP is MSP—Linker—MSP, wherelinker may be either the Linker 1 or Linker 2 sequence defined below andMSP may be any of the monomeric membrane scaffold proteins previouslydefined. Linker 1 (Lb1) is composed of 4 amino acids, preferably thesequence Asn-Pro-Gly-Thr (SEQ ID NO:104). Linker 2 (Lb2) is composed of6 amino acids with one additional residue on both ends to provide moreflexibility, preferably the sequence Ser-Asn-Pro-Gly-Thr-Gln (SEQ IDNO:136).

TABLE 42 DNA sequence encoding MSP2N4(His-TEV-H2S-H3-H4-H5-H6-H7-H8-H9-H10-NPGT- H2-H3-H4-H5-H6-H7-H8-H9-H10)(SEQ ID NO:112)      atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccagaatccaggtacccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag

TABLE 43 Amino acid sequence of MSP2N4 (SEQ ID NO:113)MGHHHHHHHDYDIPTTENLYFQGSVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQNPGTPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

TABLE 44 DNA sequence encoding MSP2N5(His-TEV-H2S-H3-H4-H4-H5-H6-H7-H8-H9-NPGT-H3-H4- H4-H5-H6-H7-H8-H9-H2)(SEQ ID NO:114)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgaatccaggtaccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtcc

TABLE 45 Amino acid sequence of MSP2N5 (SEQ ID NO:115)MGHHHHHHHDYDIPTTENLYFQGSVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLNPGTKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVTQEFWDNLEKETEGLRQEMS

TABLE 46 DNA sequence encoding MSP2N6(His-TEV-H2S-H3-H4-H4-H5-H6-H7-H8-H9-SNPGTQ-H3-H4- H4-H5-H6-H7-H8-H9-H2)(SEQ ID NO:116)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgtccaatccaggtacccaaaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtcc

TABLE 47 Amino acid sequence MSP2N6 (SEQ ID NO:117)MGHHHHHHHDYDIPTTENLYFQGSVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLSNPGTQKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVTQEFWDNLEKETEGLRQEMS

Fusion constructs of membrane scaffold proteins have been constructedwith other proteins and peptides. Fusions with cytochrome P450 reductase(CPR) include the following:

TABLE 48 DNA sequence encoding MSP2CPR (MSP2-linker-CPR, linker aminoacid sequence is VD and CPR is the rat cytochrome P450 reductasecomplete sequence) (SEQ ID NO:118)atgggtcatcatcatcatcatcacattgagggacgtctgaagctgttggacaattgggactctgttacgtctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccagggtaccctgaagctgttggacaattgggactctgttacgtctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccagtcgaccatgggagactctcacgaagacaccagtgccaccatgcctgaggccgtggctgaagaagtgtctctattcagcacgacggacatggttctgttttctctcatcgtgggggtcctgacctactggttcatctttagaaagaagaaagaagagataccggagttcagcaagatccaaacaacggccccacccgtcaaagagagcagcttcgtggaaaagatgaagaaaacgggaaggaacattatcgtattctatggctcccagacgggaaccgctgaggagtttgccaaccggctgtccaaggatgcccaccgctacgggatgcggggcatgtccgcagaccctgaagagtatgacttggccgacctgagcagcctgcctgagatcgacaagtccctggtagtcttctgcatggccacatacggagagggcgaccccacggacaatgcgcaggacttctatgactggctgcaggagactgacgtggacctcactggggtcaagtttgctgtatttggtcttgggaacaagacctatgagcacttcaatgccatgggcaagtatgtggaccagcggctggagcagcttggcgcccagcgcatctttgagttgggccttggtgatgatgacgggaacttggaagaggatttcatcacgtggagggagcagttctggccagctgtgtgcgagttctttggggtagaagccactggggaggagtcgagcattcgccagtatgagctcgtggtccacgaagacatggacgtagccaaggtgtacacgggtgagatgggccgtctgaagagctacgagaaccagaaaccccccttcgatgctaagaatccattcctggctgctgtcaccgccaaccggaagctgaaccaaggcactgagcggcatctaatgcacctggagttggacatctcagactccaagatcaggtatgaatctggagatcacgtggctgtgtacccagccaatgactcagccctggtcaaccagattggggagatcctgggagctgacctggatgtcatcatgtctctaaacaatctcgatgaggagtcaaacaagaagcatccgttcccctgccccaccacctaccgcacggccctcacctactacctggacatcactaacccgccacgcaccaatgtgctctacgaactggcacagtacgcctcagagccctcggagcaggagcacctgcacaagatggcgtcatcctcaggcgagggcaaggagctgtacctgagctgggtggtggaagcccggaggcacatcctagccatcctccaagactacccatcactgcggccacccatcgaccacctgtgtgagctgctgccacgcctgcaggcccgatactactccattgcctcatcctccaaggtccaccccaactccgtgcacatctgtgccgtggccgtggagtacgaagcgaagtctggccgagtgaacaagggggtggccactagctggcttcgggccaaggaaccagcaggcgagaatggcggccgcgccctggtacccatgttcgtgcgcaaatctcagttccgcttgcctttcaagtccaccacacctgtcatcatggtgggccccggcactgggattgcccctttcatgggcttcatccaggaacgagcttggcttcgagagcaaggcaaggaggtgggagagacgctgctatactatggctgccggcgctcggatgaggactatctgtaccgtgaagagctagcccgcttccacaaggacggtgccctcacgcagcttaatgtggccttttcccgggagcaggcccacaaggtctatgtccagcaccttctgaagagagacagggaacacctgtggaagctgatccacgagggcggtgcccacatctatgtgtgcggggatgctcgaaatatggccaaagatgtgcaaaacacattctatgacattgtggctgagttcgggcccatggagcacacccaggctgtggactatgttaagaagctgatgaccaagggccgctactcactagatgtgtggagc

TABLE 49 Amino acid sequence of MSP2CPR (SEQ ID NO:119)MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQSTMGDSHEDTSATMPEAVAEEVSLFSTTDMVLFSLIVGVLTYWFIFRKKKEEIPEFSKIQTTAPPVKESSFVEKMKKTGRNIIVFYGSQTGTAEEFANRLSKDAHRYGMRGMSADPEEYDLADLSSLPEIDKSLVVFCMATYGEGDPTDNAQDFYDWLQETDVDLTGVKFAVFGLGNKTYEHFNAMGKYVDQRLEQLGAQRIFELGLGDDDGNLEEDFITWREQFWPAVCEFFGVEATGEESSIRQYELVVHEDMDVAKVYTGEMGRLKSYENQKPPFDAKNPFLAAVTANRKLNQGTERHLMHLELDISDSKIRYESGDHVAVYPANDSALVNQIGEILGADLDVIMSLNNLDEESNKKHPFPCPTTYRTALTYYLDITNPPRTNVLYELAQYASEPSEQEHLHKMASSSGEGKELYLSWVVEARRHILAILQDYPSLRPPIDHLCELLPRLQARYYSIASSSKVHPNSVHICAVAVEYEAKSGRVNKGVATSWLRAKEPAGENGGRALVPMFVRKSQFRLPFKSTTPVIMVGPGTGIAPFMGFIQERAWLREQGKEVGETLLYYGCRRSDEDYLYREELARFHKDGALTQLNVAFSREQAHKVYVQHLLKRDREHLWKLIHEGGAHIYVCGDARNMAKDVQNTFYDIVAEFGPMEHTQAVDYVKKLMTKGRYSLDVWSFusions have been prepared with fluorescent proteins (FP) and MSPsequences. All constructs of the form His-TEV2-(FP)-MSP1T2 orHis-TEV-MSP1T2-GT-(FP), where (FP) is the enhanced green fluorescentprotein (EGFP), the enhanced yellow fluorescent protein (EYFP) or cyanfluorescent protein (CFP).

The overall N-terminal sequences are of the form: His-TEV2 (which havebeen modified to incorporate a BamH1 restriction site into thesequence). The modified His-TEV2 DNA sequence isatgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggatcc(SEQ ID NO:120), and the modified His-TEV2 Protein sequence isMGHHHHHHHDYDIPTTENLYFQGS (SEQ ID NO:121).

The fluorescent proteins have the following DNA and protein sequences:

TABLE 50 DNA sequence encoding EGFP (SEQ ID NO:122)gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag

TABLE 51 Amino acid sequence of EGFP (SEQ ID NO:123)VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGM DELYK

TABLE 52 DNA sequence encoding EYFP (SEQ ID NO:124)gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcagtgcttcgcccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag

TABLE 53 Amino acid sequence of EYFP (SEQ ID NO:125)VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGM DELYK

TABLE 54 DNA sequence encoding ECFP (SEQ ID NO:126)gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctggggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacatcagccacaacgtctatatcaccgccgacaagcagaagaacggcatcaaggccaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa

TABLE 55 Amino acid sequence of ECFP (SEQ ID NO:127)VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMD ELYK

TABLE 56 DNA sequence encoding His-TEV-MSP1T2-GT (SEQ ID NO:128)atgggtcatcatcatcatcatcatcacgattatgatattcctactactgagaatttgtattttcagggttctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccagggtacc

TABLE 57 Amino acid sequence of His-TEV-MSP1T2-GT (SEQ ID NO:129)MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTMSP derivatives have been prepared with the incorporation of cysteineresidues into the scaffold proteins by point mutation. DNA coding andamino acid sequences are given in Tables 58 and 59, respectively. InMSP1RC12′ a cysteine residue is incorporated at the last residue in theFactor X recognition site. This mutant is used to prepare fluorescentlylabeled discs and attach to surfaces or matrices. In MSP1K90C, Lysine90is replaced by a cysteine. See Tables 60 and 61 for coding and aminoacid sequences respectively. In MSP1K152C, Lysine 152 is replaced bycysteine; see Tables 62 and 63.

TABLE 58 DNA sequence encoding MSP1RC12′ (SEQ ID NO:130)Atgggtcatcatcatcatcatcacattgagggatgtctgaagctgttggacaattgggactctgttacgtctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag

TABLE 59 MSP1RC12′ Protein Sequence (SEQ ID NO:131)MGHHHHHHIEGCLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

TABLE 60 DNA sequence encoding MSP1K90C (SEQ ID NO:132)atgggtcatcatcatcatcatcacattgagggacgtctgaagctgttggacaattgggactctgttacgtctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaatgtctccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcgaaagcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag

TABLE 61 MSP1K90C Protein sequence (SEQ ID NO:133)MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQCLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

TABLE 62 DNA sequence encoding MSP1K152C (SEQ ID NO:134)atgggtcatcatcatcatcatcacattgagggacgtctgaagctgttggacaattgggactctgttacgtctaccttcagtaaacttcgcgaacaactgggccccgtgacgcaggaattctgggacaacctggaaaaagaaaccgagggactgcgtcaggaaatgtccaaagatttagaagaggtgaaggccaaggttcagccatatctcgatgactttcagaaaaaatggcaggaagagatggaattatatcgtcaaaaggtggaaccgctgcgtgcggaactgcaagagggggcacgccaaaaactccatgagctccaagagaagctcagcccattaggcgaagaaatgcgcgatcgcgcccgtgcacatgttgatgcactccggactcatttggcgccgtattcggatgaacttcgccagcgtttggccgcacgtctcgaggcgctgaaagaaaacgggggtgcccgcttggctgagtaccacgcatgcgcgacagaacacctgagcaccttgagcgaaaaagcgaaaccggcgctggaagatctacgccagggcttattgcctgttcttgagagctttaaagtcagttttctgtcagctctggaagaatatactaaaaagctgaatacccag

TABLE 63 MSP1K152C Protein sequence (SEQ ID NO:135)MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHACATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

The mutations in MSP1K90C and in MSP1K152C are located on inter-helicalinterfaces. Discs were formed in the presence of DTT. The discs are morestable toward tempe rature-induced irreversible degradation. These areour variants of the “Milano” mutations.

In addition to these sequences, there are two fusion protein constructsof reference. These are composed of two MSP1 constructs linked by aGly-Ser linker: MSP2 (MSP1-Gly-Thr-MSP1, SEQ ID NO:17) and MSP2D1D1(MSP1T3-Gly-Thr-H2-H3-H4-H5-H6-H7-H8-H9-H10, SEQ ID NO:86).

Other constructs that can be readily produced include permutations ofthe above, i.e. MSP1 or MSP2 or MSP2a with any combination of thefollowing: hinge deletion, hinge replacement, half-repeat deletion,histidine tag, different linkers for MSP2 analogs.

Example 3 Expression of Recombinant MSPs

To express MSP proteins, the nucleic acid constructs were insertedbetween the NcoI and HindIII sites in the pET28 expression vector andtransformed into E. coli BL21 (DE3). Transformants were grown on LBplates using kanamycin for selection. Colonies were used to inoculate 5ml starter cultures grown in LB broth containing 30 μg/ml kanamycin. Foroverexpression, cultures were inoculated by adding 1 volume overnightculture to 100 volumes LB broth containing 30 μg/ml kanamycin and grownin shaker flasks at 37° C. When the optical density at 600 nm reached0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to aconcentration of 1 mM to induce expression and cells were grown 3-4hours longer before harvesting by centrifugation. Cell pellets wereflash frozen and stored at −80° C.

Example 4 Purification of Recombinant MSPs

Purification of histidine-tagged MSPs was carried out as follows. Afrozen cell pellet from 1 liter of expression culture was resuspended in25 milliliters of 20 mM Tris HCl pH 7.5 containing 1 mMphenylmethylsulfonyl fluoride. Triton X-100(t-octylphenoxypolyethoxyethanol) was added from a 10% (w/v) stock indistilled H20to a final concentration of 1%. The resuspended cells weresonicated on ice at 50% duty cycle at a power setting of 5 for fourcycles of 1 minute on, 5 minutes off with a Branson probe sonifier. Theresulting lysate was centrifuged for 30 minutes at 30,000 rpm in aBeckman Ti 45 rotor in a ultracentrifuge. The resulting supernatant wasfiltered through a 0.22 μm nylon syringe filter. The salt concentrationwas adjusted to 0.5 M from a 4 M NaCl stock in water and applied to a 5ml Hi-Trap nickel loaded column (Pharmacia, Piscataway, N.J.).

For His-tagged-MSP1, the column is washed with 20 ml buffer (10 mM TrispH 8, 0.5 M NaCl) containing 1% Triton X-100, followed by 20 mlbuffer+50 mM sodium cholate, and then 20 ml buffer and 20 ml 100 mMimidazole in buffer. The His-tagged polypeptide is eluted with 15 ml 0.5M imidazole in buffer.

For His-tagged-MSP2, the column is washed with 20 ml buffer (10 mM TrispH 8, 0.5 M NaCl) containing 1% Triton X-100; 20 ml buffer+50 mMcholate; 20 ml buffer; 20 ml 35 mM imidazole in buffer. The His-taggedpolypeptide is then eluted with 15 ml 0.5 M imidazole in buffer, and thepurified protein is dialyzed against 10 mM Tris pH 8, 0.15 M NaCl usinga 10,000 MW cutoff cellulose dialysis membrane.

Example 5 Production of MSP-containing Nanoscale Particles

To reconstitute MSP proteins of the present invention with lipid,purified MSP was concentrated in a pressurized ultrafiltration device(Amicon) using a 10,000 MW cutoff filter to ˜2-6 mg protein/ml.Concentration of protein was determined by bicinchoninic acid assay(Pierce Chemical, Rockford, Ill.) or measurement of A280 usingtheoretical absorption coefficient. Phospholipid (dipalmitoylphosphatidylcholine in this case, however different phosphatidylcholinesand mixtures of phosphatidylcholine and other lipids can be used) inchloroform stock solution was dried under a stream of nitrogen andplaced in vacuo overnight. Phosphate analysis was performed to determinethe concentration of chloroform stock solutions. The dried lipid filmwas resuspended in buffer 10 mM Tris HCl pH 8.0 or pH 7.5 containing0.15 M NaCl and 50 mM sodium cholate to give a final lipid concentrationof 25 mM. The suspension was vortexed and heated to 50° C. to obtain aclear solution. Phospholipid solution was added to solution of MSP (2-6mg/ml protein) to give molar ratios for MSP1 :lipid of 2:200 and forMSP2 of 1:200. The mixture was incubated overnight at 37° C. and thendialyzed against 1000 volumes of buffer without cholate with 4 changesof buffer over 2-3 days.

Example 6 Tissue Factor Incorporation

Tissue Factor (TF) is a representative membrane protein. In order todemonstrate the value of MSP technology for a tethered membrane protein,recombinant human TF was incorporated into MSP-supported Nanodiscs. Therecombinant protein consists of an extracellular domain, thetransmembrane anchor and a truncated cytosolic domain. The truncationincreases the homogeneity of the protein by removing the C-terminalportions of the protein which are subject to proteolysis by bacterialenzymes. This modification does not affect TF activity. Additionalmodifications to the protein include an N-terminal trafficking peptideand an HPC4 epitope tag. The trafficking peptide directs the expressedprotein to the intermembrane space of the recombinant E. coli host cell,in which space the peptide sequence is cleaved. The HPC4 epitope allowsfor affinity purification with Ca²⁺ dependent antibody (Rezaie et al.,1992) and does not affect TF activity.

A 25 mM lipid mixture containing 80% phosphatidyl choline and 20%phosphatidyl serine was solubilized with 50 mM cholate in 10 mM Tris Cl,150 mM NaCl at pH 8.0. TF, MSP1 and lipid (in a ratio of 1:10:1000) werecombined and incubated overnight at 37° C. The sample was then dialyzedat 37° C. (10,000 dalton molecular weight cutoff membrane) againstbuffer containing 10 mM Tris Cl, 150 mM NaCl at pH 8.0 (lacking cholate)for 2 hours. Dialysis was then continued at 4° C. for an additional 6hours with buffer changes every 2 hours. The approximately 1 ml samplewas then concentrated to <250 μl using a YM-10 centrifuge concentratorand injected into a Pharmacia 10/30 Superdex 200 HR gel filtrationcolumn. Samples were eluted with buffer identical to that describedabove (no cholate) at 0.5 ml per minute. Fractions from chromatographywere run on an 8-25% gradient SDS polyacrylamide gel to determineapparent size and then checked for coagulation activity. Thechromatogram showing elution of TF incorporated into an excesspopulation of MSP1 Nanodiscs is shown in FIG. 16A-16B.

The activity of TF in several disk fractions was determined bycoagulation assays with human serum. Activity was monitored in fractions25-28 as the inverse of coagulation time. Activity was highest infraction 25 at 40 hr⁻¹ and decreased through fraction 28 at 30 hr⁻¹.This is expected from the size chromatogram in that the leading edge ofthe Nanodisc peak has a larger effective mass due to the incorporationof TF in the MSP-supported bilayer. This assay thus demonstrates that TFis incorporated into Nanodiscs in an active conformation and that themembrane environment of the Nanodisc closely mimics that of the nativemembrane system.

Cytochrome b5 is a membrane anchored heme protein having a singlemembrane anchor domain that penetrates the membrane bilayer. Cytochromeb5 solubilized from its native membrane exists as large aggregates inthe absence of detergent and appears as a smear rather than a discreteband on native polyacrylamide gel electrophoresis. Formation ofNanodiscs through a self-assembly process wherein cytochrome b5 is addedto the preparation of MSP and phospholipid results in the incorporationof cytochrome b5 into Nanodisc structures. This is verified by theintense heme staining of the band corresponding to Nanodiscs. The datashow that cytochrome b5 can be successfully solubilized using MSPtechnology and that disc complexes containing cytochrome b5 can bechromatographically separated and purified away from the undesiredaggregated material. The optical absorption properties of the hemechromophore of the purified material demonstrate that the heme activesite in a native conformation.

Nanodiscs can also be formed by mixing 20 μl of MSP1 (10 mg/ml), 6.6 μlcytochrome b5 (0.5 mM) and 50 μl egg phosphatidylcholine/sodium cholate(11.2 egg PC, 6.2 mg/ml sodium cholate), incubating overnight at 4° C.,followed by dialysis to remove cholate. Purification was accomplishedusing a Pharmacia MonoQ FPLC anion exchange column equilibrated in 25 mMTris Cl, pH 8.0. A linear gradient was run at 0.5 ml/min from 0-1 M NaClin 20 min.

As an alternative to incorporating tethered membrane proteins intoNanodiscs from solubilized, purified proteins, the tethered membraneproteins can be incorporated into Nanodiscs with MSPs using membrane ormembrane fragment preparations containing those tethered membraneproteins of interest.

Example 7 Embedded Membrane Protein Incorporation

Cytochrome P450 2B4 from rabbit liver microsomes, cytochrome P450 3A4found in nature in human liver microsomes and cytochrome P450 6B1 frominsect microsomes are representative of embedded membrane proteins.

Cytochrome P450 2B4 was isolated from rabbit liver microsomes afterinduction with phenobarbital. Formation of 2B4 Nanodiscs is as follows.Cytochrome P450 2B4 was reconstituted into disks by the detergentdialysis method. The buffer consisted of 10 mM Tris-HCl pH 8.0, 0.1 MNaCl, 10% (v/v) glycerol. The mixture of apo A-I, cholate andphospholipid (1:220:110 mole ratio) was incubated for 8 hours at 37° C.followed by addition of P450 (1:0.5, apo A-I:P450 mole ratio) andincubation overnight at room temperature. The mixture was dialyzed usinga 10,000 MW cutoff slide-a-lyzer (Pierce Chemical Co., Rockford, Ill.)at room temperature for two hours followed by a change of buffer andcontinued dialysis at 4° C. It was found that 82% of the P450 contentcould be recovered under these conditions. After dialysis, the mixturewas injected onto a Superdex 200 HR10/30 gel filtration column(Pharmacia, Uppsala, SE) equilibrated in reconstitution buffer at roomtemperature at a flow rate of 0.25 ml/minute with collection of 0.5 mlfractions. Fractions were assayed using native polyacrylamide gradientgel electrophoresis on 8-25% gradient native gels and Coomassie stainingusing the Phastgel system (Pharmacia, Uppsala, Sweden).

Human cytochrome P450 3A4, normally from liver microsomes, has also beencloned, expressed in E. coli, purified and incorporated intoMSP-supported bilayer Nanodiscs. Ten nanomoles of MSP2, one micromole oflipid, five nanomoles of cytochrome P450 3A4 protein and two micromolescholic acid were incubated together at 37° C. for 2 hours. The incubatedmixture was then dialyzed in a 10K Slide-A-lyzer Dialysis Cassette(Pierce Chemical Co., Rockford, Ill.). The dialysis was carried out with10 mM potassium phosphate (pH 7.4) 150 mM NaCl buffer. The sample wasdialyzed at 37° C. for 6 hours followed by a buffer change, and dialysiscontinued at 4° C. with two buffer changes at 12 hour intervals. Thesamples were then fractionated on a Superdex 200 HR 10/30 column(Pharmacia, Uppsala, SE) equilibrated in dialysis buffer at roomtemperature at a flow rate of 0.5 ml/min.

Cytochrome P450 6B1 is another model embedded membrane protein; it hasbeen isolated from Papilio polyxenes, the black swallowtail. Thesebutterflies feed exclusively on plants producing furanocoumarins, plantmetabolites that are phototoxic to most organisms. Cytochrome 6B1catalyzes the detoxification of furanocoumarins.

In order to show the utility of the MSP methodology of the presentinvention, we demonstrated that isolated membranes containing theirrepertoire of membrane proteins and natural lipids could be used as asource for incorporating membrane proteins into Nanodiscs. An importantillustrative embodiment is the use of the common insect cell(Sf9)-baculovirus expression system which is used widely as aheterologous expression system. Thus, we used an insect cell lineco-infected such that a microsomal preparation containing overexpressedinsect CYP6B1 and also overexpressed insect NADPH cytochrome P450reductase. In these experiments we not only demonstrate that MSPNanodiscs can be used to incorporate another cytochrome P450 system intosoluble monodisperse particles but also that the source of this P450could be simply whole membranes containing this protein.

A standard baculovirus expression system was used to obtain microsomalpreparations with overexpressed insect cytochrome CYP6B1 and insectNADPH P450 reductase. Construction of the recombinant CYP6B1 baculovirusexpression vector and infection of Spodoptera frugiperda (Sf9) wasperformed as previously described (Chen et al., 2002). Typically, 32plates containing 6×10⁷ baculovirus-infected cells each (MOI of 2) werecollected 72 hours post-infection. Microsomal membranes were homogenizedin 2 ml grinding buffer (pH 7.8) composed of 0.1 M sodium phosphatebuffer (pH 7.8), 1.1 mM EDTA, 20% glycerol, 0.5 mM PMSF, 0.1 mM DTT, and5 μg/ml (w/v) leupeptin. Membranes were frozen in liquid nitrogen andstored at −80° C.

To assemble Nanodiscs comprising CYP6B1 from the microsomal membranepreparation, the protein concentration of the membranes was determinedusing a BCA™ protein assay kit from Pierce (Rockford, Ill.). We assumeda 1:1 mass relationship of protein: lipid in the membranes and anaverage molecular weight of phospholipids of 750 grams/mole. Themembranes were detergent-solubilized with 0.5 M cholic acid(neutralized) and mixed with MSP in the approximate ratio of 1:25:50 to1:2000:1000, preferably 1:75:150 in at least some cases, forMSP:lipid:detergent. Typically, reconstitution samples includeapproximately 100 nmol scaffold protein, 10 μmol lipid, and 20 μmolneutralized cholic acid and were pre-incubated for 1.5 hours at 4° C.The temperature chosen is higher than the phase transition temperaturefor the lipids. Detergent was removed by incubating with Biobeads® SM-2Adsorbent from BioRad Laboratories (Hercules, Calif.) (0.4 gramsBiobeads per 1 ml of reconstitution mixture) for 1.5 hours at 4° C.followed by centrifugation at 11,750×g for 5 minutes. His6-tagged MSPparticles were purified by incubating with 1 ml of Ni-NTA agarose fromQiagen, Inc. (Valencia, Calif.) per 7.5 mg of His6-tagged MSP for 1 hourat 4° C., followed by centrifugation at 11,750×g for 5 minutes. MSPparticles bound to the Ni-NTA agarose were washed with three sequentialresin volumes of 0.1 M sodium phosphate buffer (pH 7.4) containing 0.3 MNaCl, 0.15 M NaCl, and no NaCl, respectively. To maintain the integrityof the CYP6B1 protein, MSP particles were eluted with 0.1 M sodiumphosphate buffer (pH 7.4) containing 0.25 M EDTA (to chelate trace metalions) rather than the 50 mM imidazole used in previous MSPpurifications.

Based on the lipid concentration contained in the microsomalpreparations, MSP technology was used to assemble microsomal proteinsinto nanoparticle discs using a ratio of 110:1:220 lipid:MSP1:cholate.The microsomal sample was detergent solubilized with neutralized cholateand mixed with MSP1. The sample was incubated at 4° C. for 2 hours. Thedetergent can be removed by dialysis or adsorption to hydrophobic beads.In this experiment Biobeads (hydrophobic beads, trademark of BioRad,Hercules, Calif.) were added in excess (0.25 g per 1 ml disc mixture)and incubated for 2 hours at 4° C. for 2 hours to remove detergent. Thesample was removed from the beads and the His₆-tagged MSP was isolatedby using a batch purification method with Ni²⁺ resin. The MSP disks werethen isolated by Superdex sizing column chromatography (FIG. 9).Incorporation of P450 into the His₆-tagged discs was followed by COdifference spectroscopy of nickel affinity column purified and sizingcolumn-purified fractions (FIG. 10). SDS-PAGE was performed using 8-25%gradient gels stained with Coomassie blue to verify incorporation ofcytochrome P450 6B1 into discs (FIG. 10).

The endogenous (natural) ratio of cytochrome P450 to reductase is about10-20. To obtain activity of the cytochrome P450 6B1 afterreconstitution into discs, it is preferred that an excess of reductasebe added to the reconstitution mixture, such that a P450 molecule andreductase molecule both partition into a single disc. Supplementation ofthe microsomal preparation with exogenously added reductase has beensuccessfully demonstrated.

The protocol for making discs using microsomal preparations was usedwith one modification. Exogenous rat reductase was added after thesolubilization step of the microsomal preparation with sodium cholateand before the addition of MSP1. Otherwise identical disc assembly andpurification procedures were followed. The sample was separated by aSuperdex sizing column, where absorbance at 280 nm indicates thepresence of MSP1, absorbance at 420 and 456 nm indicates the presence offerric species, and absorbance at 456 nm also indicates presence ofreductase. A ratio plot of 456 to 420 nm was made; it showed positionson the chromatogram where the absorbance at 456 nm was above thatassociated with cytochrome P450 6B1 and, therefore, could be attributedto absorbance by reductase. Retention times reflected the presence of 10nm particles containing cytochrome P450 6B1 and reductase (FIG. 13).

MSP-supported Nanodiscs with purified proteins, membrane fragments ordisrupted membranes can be used in high throughput screening ventures,for example, to identify new pharmaceuticals and other biologicallyactive molecules.

Example 8 Integral Membrane Protein Incorporation

Bacteriorhodopsin (BR) is a model integral membrane protein, and a modelseven transmembrane domain protein. BR was incorporated into nanoscalestructures using the following procedure, which is a protocol useful forother proteins as well. BR was obtained as lyophilized purple membranefrom Sigma (St. Louis, Mo.). 1 mg BR was suspended in 1 ml 25 mMpotassium phosphate, pH 6.9. 1 ml 90 mM n-octyl β-D-glucopyranoside inthe same buffer was added and the sample placed in the dark at 24° C.overnight. This treatment produces a detergent-solubilized monomericform (Dencher et al., 1982). BR was quantitated assuming a molarextinction coefficient at 550 nm of 63,000. BR (7.8 μM) was mixed withMSP1 (97 μM) or MSP2 (110 μM) and cholate (50 mM) to give final molarratios of MSP1:BR of 10:1 or MSP2:BR of 5:1 and a cholate concentrationof approximately 8 mM. For reconstitution with phospholipid, the lipidis solubilized as above in the presence of 50 mM cholate and mixed withMSP1 at a mole ratio of 1 MSP1:110 lipids:0.1 BR. The mixture wasincubated at room temperature for ˜3 hours followed by dialysisovernight against 1000 volumes of buffer using 10,000 MW cutoff dialysisdevices (Slide-a-lyzer, Pierce Chemical). Dialysis was continued at 4degrees for 2 days with several changes of buffer. 10 mM HEPES, pH 7.5,0.15 M NaCl buffer can be used. Tris buffer pH 7.5 or pH 8 has also beenused successfully.

The human 5-hydroxytryptamine 1A G protein coupled receptor has beenincorporated into MSP-containing nanoparticles. A commercially availableinsect cell expression system that provides a membrane fractioncontaining the human 5-hydroxytryptamine 1A (5-HT-1A) GPCR was used as asource of this GPCR to prepare Nanodiscs. Briefly, the 5-HT-1A receptorcontaining membrane preparation was mixed with phospholipids(phosphatidyl choline, phosphatidylethanolamine, phosphatidyl serine) ata ratio of 45:45:10, MSP1 and cholate (neutralized cholic acid).

5-HT-1A receptors overexpressed in a commercially available Sf9 insectcell membrane preparation (Sigma Chemical Co., St. Louis, Mo.) weresolubilized using the following protocol. POPC, POPS and POPE (AvantiPhospholipids) in chloroform were mixed in a 45:10:45 mole ratio anddried down under a stream of nitrogen, then placed under vacuum forseveral hours to remove residual solvent. The phospholipids weredispersed in 50 mM Tris pH 7.4, 0.2 M NaCl, 50 mM sodium cholate bufferat a concentration of 25 mM phospholipid. Five microliters of the Sf9membrane preparation (0.2 mg/ml protein), 1.62 microliters ofphospholipid in buffer, 2.4 microliters of MSP1 (4.2 mg/ml) and 0.28microliters 4 M NaCl were mixed and left for 1 hour on ice. The mixturewas diluted to 100 microliters total volume with 50 mM Tris pH 7.4 anddialyzed in a mini slide-a-lyzer (Pierce Chemical) against 50 mM Tris pH7.4 at 4° C. (two one-liter changes of buffer).

To determine the amount of 5-HT-1A receptor associated with Nanodiscs, aradiolabeled ligand was bound to the receptor and disk-receptor-ligandcomplexes were isolated using the 6-histidine tag present in the MSP1according to the following protocol. After dialysis, the mixture wasdiluted to 200 microliters total volume with 50 mM Tris pH 7.4.Ninety-five microliters of the diluted mixture were placed into each oftwo tubes. One hundred five microliters of stock reagent were added togive final concentrations of 50 mM Tris pH 7.4, 10 mM MgSO₄, 0.5 mMEDTA, 0.1% ascorbic acid in a final volume of 200 microliters.Tritium-labeled 8-hydroxy-DAT (specific activity 135000 Ci/mole) wasadded to each tube to give a concentration of 1.5 nM. As a control,unlabeled metergoline (final concentration 100 micromolar) was added toone of the tubes as a competitive ligand. After 1 hour on ice, themixture was applied to 200 microliters of Ni-chelating resin tospecifically bind receptor associated with His-tagged MSP1 disks. Theresin was washed three times with 0.5 ml of cold 50 mM Tris pH 7.4 toremove non-specifically bound ligand. Specifically bound radiolabeled8-hydroxy-DAT bound to receptor/disk complexes was eluted with 0.5 ml0.5 M imidazole in 10 mM Tris pH 7.4, 0.5 M NaCl. Scintillation cocktailwas mixed with the eluate and specifically bound radioligand wasdetermined by scintillation counting. Between five and fifteen percentof the receptor initially present in the Sf9 membrane was found to bindligand in receptor associated with MSP1 Nanodiscs.

The particles into which the 5-HT-1A GPCR had incorporated weredialyzed. Functionality (in terms of ligand binding) was tested byincubation with buffer containing tritiated 8-OH-DAT, an agonist of thisreceptor. The particles were then run over a Ni-NTA column to bind viathe histidine tag on the MSP1 and to separate the particles from8-OH-DAT which had not bound to the particles, and the material bound tothe column was then eluted. Association of the tritium labeled agonistwas demonstrated, showing that the incorporated GPCR retained itsability to bind agonist.

As discussed above for the tethered membrane proteins, the integral andembedded membrane proteins can be incorporated into Nanodiscs using MSPsand solubilized membrane preparations, rather than purified, solubilizedproteins. The naturalistic presentation of the proteins within theNanodiscs is maintained, regardless of whether the proteins werepurified or whether they were directly derived from membranepreparations.

Example 9 Analysis of MSP-Supported Nanodisc Phospholipid Assemblies

The particles resulting from self-assembly of membrane scaffold proteinsand phospholipids, either with or without an additional target protein,were analyzed as follows.

Bacteriorhodopsin-containing particles were dialyzed, and the resultingmixture was injected onto a Superdex 200 HR10/30 gel filtration column(Pharmacia) and eluted with buffer at 0.5 ml/min at room temperature.Absorbance was monitored at 280 nm for protein and 550 nm for BR. 0.5 mlfractions were collected. The column was calibrated using a mixture ofthyroglobulin (669 kDa, Stoke's diameter 170 A), ferritin (440 kDa,Stoke's diameter 122 A), catalase (232 kDa, Stoke's diameter 92 A),lactate dehydrogenase (140 kDa, Stoke's diameter 82 A), bovine serumalbumin (66 kDa, Stoke's diameter 71 A), and horse heart cytochrome c(12.4 kDa, Stoke's diameter 35.6 A).

Atomic Force Microscopy was performed with a Digital InstrumentsNanoscope IIIa in contact mode with sharpened silicon nitride probesunder buffer. MSP1 and MSP2 dipalmitoyl phosphatidylcholine particleswere treated with 1:50 Factor Xa:MSP protein by mass in 10 mM Tris pH 8,0.15 M NaCl, 2 mM CaCl₂ for 8 hours. 2-10 ml sample was placed on afreshly cleaved mica surface along with 20 ml imaging buffer (10 mM TrispH 8, 0.15 M NaCl, 10 mM MgCl₂) and incubated for 30 minutes or longerbefore mounting sample in the fluid cell. Several milliliters of bufferwere flushed through the fluid cell to remove unadsorbed material.

Phosphate analysis of the nanoscale particles was carried out asfollows. Phosphate assay procedures were adapted from Chen et al. (1956)and Fiske and Subbarow (1925). Samples containing roughly 40 nmoleslipid phosphate were dried down in glass tubes. 75 μl 8.9 N H₂SO₄ wasadded to each tube and heated to 210° C. for 30 minutes. 1 drop 30% H₂O₂was added to each tube and heated for 30 minutes. Tubes were cooled,0.65 ml H₂O was added followed by 83.3 μl 2.5% w/v ammonium molybdatetetrahydrate followed by vortexing and the addition of 83.3 μl 10% w/vascorbic acid. After mixing, the tubes were placed in a boiling waterbath for 7 minutes. Absorbance was read at 820 nm. Absorbance wascalibrated using potassium phosphate standards from 0 to 100 nmolphosphate. Buffer blanks from column chromatography were included forMSP proteins.

Example 10 MSP-supported Structures on Surfaces

Nanodiscs comprising MSPs and a protein of interest can be assembledonto a gold surface or other solid surface (solid support). The utilityof this relates to the resulting epitaxial presentation of a targetincorporated into a Nanodisc assembly to the solution. This offers anideal system for quantitating binding of other macromolecules or smallmolecules tagged with dielectric contrast agents to the target protein.A common method of accomplishing such measurements uses surface plasmonresonance (SPR) technology. SPR is a common technique used to monitorbiomolecular interactions at surfaces. The ability of SPR to rapidlydetect and quantitate unlabeled protein interactions on gold surfaces isuseful for creating high through put chip assays for diverse membraneproteins (embedded and solubilized) on discs.

Discs consisting of the phospholipid DPPC either with or without anadditional thiolated lipid and MSP1 protein were prepared as follows. A25 mM lipid mixture containing phosphatidylcholine was solubilized with50 mM cholate in 10 mM Tris Cl, 150 mM NaCl at pH 8.0 were combined andincubated overnight at 37° C. For thiolated discs, 90%phosphatidylcholine and 10% thiolated lipid (ATA-TEG-DSPA, NorthernLipids, Vancouver, BC, Calif.) was solubilized in 3.3 mM Tris Cl, 66.7mM borate, 150 mM NaCl at pH 9.0 in order to unmask the thiols in thethiolated lipids. MSP1 and lipid (1:100) were combined and incubatedovernight at 37° C. The sample was then dialyzed at 37° C. (10,000 MWcutoff membrane) against buffer containing 10 mM Tris Cl, 150 mM NaCl atpH 8.0 without cholate for 2 hours. Dialysis was then continued at 4° C.for an additional 6 hours with buffer changes every 2 hours. Theapproximately 1 ml sample was concentrated to <250 μl using a YM-10centrifuge concentrator and injected onto a Pharmacia 10/30 Superdex 200HR gel filtration column. Samples were eluted from the column using thestated buffer without cholate at flow rates of 0.5 ml/min. Fractionsfrom chromatography were analyzed by polyacrylamide gel electrophoresisusing 8-25% gradient polyacrylamide gel to determine apparent size.

The Nanodisc samples (3-20 μM) prepared as described were injected intoan SPR instrument to determine if the discs would bind to the goldsurface. Both the DPPC and 10% thiolated lipid discs adsorbed to a goldsurface and a modified gold surface covered with a monolayer of methylterminated thiol (nonanethiol) or carboxyl terminated thiol(11-mercaptoundecanoic acid). Thiolated discs were injected using abuffer consisting of 3.3 mM Tris, 66.7 mM borate, 150 mM NaCl, pH 9.0.DPPC discs were injected using a buffer of 10 mM Tris, 150 mM NaCl, pH7.5 or pH 8.0. In all cases, the discs could not be removed even underharsh conditions (0.5 M HCl). Surface coverage was shown to increasewith increasing concentration of discs injected (3 μM vs. 19 μM). Discsdo not form perfectly packed monolayers; accordingly, surface coverageis limited by the jamming limit (theoretical maximum coverage based onrandom sequential absorption to the surface modeling discs as identicalnon-overlapping hard spheres) of 0.547. The coverage for a fullmonolayer of discs was calculated based on an assumption of disc heightof 5.5 nm and a refractive index between 1.45 and 1.5. The fullmonolayer values were multiplied by the jamming limit to determine themaximum coverage that was then used to determine percent coverage basedon experimental values. When the disc concentration was at least 10 μM,the estimated coverages were between about 62 and about 103%. Theresultant SPR trace demonstrating association of the Nanodiscs to thegold surface is shown in FIG. 14.

Nanodiscs comprising MSPs and a protein of interest can be attached to asolid support via the His tag on the MSP where the support is coatedwith Ni-NTA or a His tag-specific antibody, commercially available fromBD Biosciences Clontech, Palo Alto, Calif., for example, or to Ni-NTAagarose beads, commercially available from Qiagen, Valencia, Calif., forexample, or other solid support, including beads, chips, plates andmicrotiter dishes.

Example 11 General Techniques

For SDS-PAGE, microliter samples were separated on 8-25% gradientpolyacrylamide gels (Pharmacia) and stained with Coomassie blue.

Sizing column chromatography purification was carried out as follows.The nickel affinity-purified sample mixture was injected onto a Superdex(Trademark of Pharmacia, Piscataway, N.J.) 200 HR10/30 gel filtrationcolumn (Pharmacia) equilibrated in 0.1M sodium phosphate buffer (pH 7.4)at a flow rate of 0.5 ml/min. Fractions containing CYP6B1 wereconcentrated using a Centricon YM-30 centrifugal filter device(Millipore Corporation, Billerica, Mass.) and re-injected onto theSuperdex 200 HR10/30 gel filtration column under the same bufferconditions.

Lipids were extracted by the Folch method (Folch-Pi et al. (1957)),where the sample was homogenized with 2:1 chloroform-methanol (v/v) andwashed with ¼ volume 0.88% KCl in water. The solution was mixedvigorously and the phases were completely separated by centrifugation(3,000×g) for 5 minutes.

Nanodisc assembly is generally carried out as follows. The proteinconcentration of the membranes was determined using a BCA™(bicinchoninic acid) protein assay kit from Pierce (Rockford, Ill.). Weassumed a 1:1 mass relationship of protein: lipid in the membranes withan average molecular weight of phospholipids of 750 grams/mole. Themembranes were detergent solubilized with 0.5 M cholic acid(neutralized) and mixed with MSP in the approximate ratio of 1:25:50 to1:2000:1000 with 1:75:150 preferable. The membranes were detergentsolubilized with 0.5 M cholic acid (neutralized) and mixed with MSP inthe approximate ratio of 1:100:200 for MSP:lipid:detergent. Typically,reconstitution samples include approximately 100 nmol membrane scaffoldprotein, 10 μmol lipid, and 20 μmol cholate and were pre-incubated for1.5 hours at 4° C. Detergent was removed by incubating with Biobeads®SM-2 Adsorbent from BioRad Laboratories (Hercules, Calif.) (0.4 gramsBiobeads per 1 ml of reconstitution mixture) for 1.5 hours at 4° C.followed by centrifugation at 11,750×g for 5 minutes. His6-tagged MSPparticles were purified by incubating with 1 ml of Ni-NTA agarose fromQiagen, Inc. (Valencia, Calif.) per 7.5 milligrams of His6-tagged MSPfor 1 hour at 4° C., followed by centrifugation at 11,750×g for 5minutes. MSP particles bound to the Ni-NTA agarose were washed withthree sequential resin volumes of 0.1 M sodium phosphate buffer (pH 7.4)containing 0.3 M NaCl, 0.15 M NaCl, and no NaCl, respectively. Tomaintain the integrity of the CYP6B1 protein, MSP particles were elutedwith 0.1 M sodium phosphate buffer (pH 7.4) containing 0.25 M EDTArather than the 50 mM imidazole used in previous MSP purifications.

Thin-Layer Chromatography (TLC) is carried out as follows. Samples werespotted onto preparative silica gel stationary phase TLC platespurchased from EM Science (Hawthorne, N.Y.) alongside phospholipidstandards purchased from Avant (Alabaster, Ala.) and developed using amobile phase of chloroform/methanol/ammonium hydroxide (65:25:4). TLCplates were exposed to iodine vapor for visualization, scanned using aHewlett Packard ScanJet, and quantified on a Macintosh computer usingthe public domain NIH Image program developed at the U.S. NationalInstitutes of Health (available on the internet at the website entitledrsb.info.nih.gov/nih-image).

Example 12 Substrate Binding

The CYP6B1-containing population of Nanodiscs collected after Superdexsize fractionation was concentrated to an enzyme concentration of 50 nM.A microtiter plate was arranged with wells A1-A5 and wells B1-B5 eachcontaining 200 μl Nanodisc samples and wells C1-C5 each containing 200μl buffer (0.1 M sodium phosphate, pH 7.4). To rows A and C, a 20 mMstock concentration of xanthotoxin (Sigma Chemical Co.) in methanol wasadded to yield final concentrations of 0 μM (column 1), 10 μM (column2), 20 μM (column 3), 50 μM (column 4), and 150 μM (column 5). Thisdilution was such that the total organic solvent content did not exceed1% when added to the Nanodisc samples. To row B, 0 μl, 0.1 μl, 0.2 μl,0.5 μl, and 1.5 μl methanol were added.

The contents of each microtiter well were scanned at 1 nm incrementsusing a SpectraMAX® Plus microplate spectrophotometer (MolecularDevices, Sunnyvale, Calif.) and were corrected for the background bufferabsorbance (defined in row C) and Nanodisc absorbance (well A1).

Example 13 Nanodiscs with Larger MSPs

The relatively larger Nanodiscs (the “extended” membrane scaffoldprotein sequences) are useful in controlling the oligomerization stateof 7-Tm receptors or other hydrophobic or partially hydrophobic proteinswhich are particularly large or which tend to oligomerize incorporatedinto Nanodiscs. As specifically exemplified, a bacteriorhodopsin trimeris self-assembled in larger nanodiscs using the longer MSPs.

Purple membrane was isolated from Halobacterium salinarum JW-3 culturesas described (Oesterhelt and Stoeckenius 1974). Sucrose was removed bycentrifugation at 35,000 rpm in a Beckman Ti-45 rotor for 15 minutesfollowed by resuspension in water. This process was repeated threetimes, the sample was aliquoted, lyophilized and stored at −20° C.Concentrations of MSPs were determined from absorbance at 280 nm usingextinction coefficients of 24740 M⁻¹ cm⁻¹ for MSP1 and 31720 M⁻¹ cm⁻¹for the other MSPs based on calculated extinction coefficient (Gill andvon Hippel 1989). The extinction coefficient in nanodisc buffer forMSP1E1, MSP1E2 and MSP1E3 was found to be equal to the calculated valuein 20 mM phosphate buffer, 6 M guanidine HCl pH 6.5. DMPC was obtainedfrom Avanti Polar Lipids, Inc., dissolved in chloroform and quantitatedby phosphate analysis (Chen, Toribara et al. 1956). Buffer consisted of10 mM Tris HCl pH 7.4, 0.1 M NaCl, 0.01% NaN₃ unless stated otherwise.Water was purified with a Milli-Q system (Millipore). All othermaterials were high-quality reagents.

To self-assemble nanodiscs with bacteriorhodopsin and extended MSPs,bacteriorhodopsin was initially solubilized with 4% w/v Triton X-100 asdescribed (Dencher and Heyn 1978). MSP stock solutions (200-400 μM) anda DMPC/cholate mixture (200/400 mM in buffer) were added to bR ineppendorf tubes or Falcon tubes (typically about 190 μM) to give MSP tobR molar ratios of 2:3 and different phospholipid ratios. After one hourat room temperature, detergent was removed by treatment for 3-4 hourswith 400 mg wet Biobeads SM-2 (BioRad) per ml of solution, with gentleagitation to keep the beads suspended (Levy, Bluzat et al. 1990). Beadswere removed by centrifuging the suspension through a pinhole made inthe bottom of the tubes.

Self-assembled Nanodisc mixtures were filtered through 0.22 micronfilters and injected onto a size exclusion chromatography column(Superdex 200 HR 10/30 column) run at 0.5 ml/min at room temperaturewith collection of one minute fractions. Peak elution was monitored at280 and 560 nm.

Nanodiscs were analyzed by SDS-PAGE, protein was quantified, and lipidstoichiometry was determined. Samples containing MSP1E3 and differentamounts of bR as calibration standards were separated on 20% SDS-PAGEusing a Phastgel system (Pharmacia) along with gel-filtration purifiedsamples of MSP1E3-bR nanodiscs. After staining with Brilliant blueR-250, gels were scanned and bands quantitated using the computerprogram NIH image to determine the ratio of bR to MSP1E3 in nanodiscs.The amount of lipid per bR in MSP1E3 disks was determined using theextinction coefficient ε₅₆₀=56,600±1200 M⁻¹ cm⁻¹ for bR in MSP1E3nanodiscs measured by the method of retinal titration and phospholipidcontent was determined by phosphate analysis (Chen, Toribara et al.1956; Rehorek and Heyn 1979). Circular dichroism spectra were measuredwith a Jasco J-720 spectrapolarimeter at ambient temperature at a sampleOD of approximately 2.

bR-MSP mixtures at a 3 to 2 ratio were titrated with lipid to determineoptimal ratios for bR solubilization as assessed by gel filtrationchromatography. The optimal ratio is chosen as the ratio at which themain peak of solubilized bR is the major component with a minimum amountof larger species. At less than optimal ratios, species of smaller sizeappear. The optimal ratios for MSP1, E1, E2, and E3 determined in thismanner are 10:1, 10:1, 55:1 and 80:1, respectively with main peak beingapproximately 80% of total bR injected. The results of reinjection ofthe pooled main peaks are shown in FIG. 19. The sizes based oncalibration of the column with a set of standard proteins are 11, 11.4,12.2, 12.8 nm in diameter for MSP1EI, MSP1E2, and MSP1E3, respectively.

bR in purple membrane exhibits excitonic interactions between bR retinalchromophores in trimers which give rise to a positive and a negativepeak in the CD spectrum. The monomeric forms of bR show a singlepositive peak arising from interaction of retinal with the proteinenvironment. CD spectra of bR solubilized by MSP's at the optimal lipidratios are shown in FIG. 20. Only MSP1E2 and MSP1E3 have a negative peakat 600 nm, indicating assembly of a trimeric form of bR in the nanoscalediscoidal particles.

Example 14 Amphotericin B-loaded Nanodiscs

Two Nanodisc preparations, one containing Amphotericin B (AmB) and onewithout (as control) are made. The ratio of MSP1T2/POPC/AmB in AmBparticles is 2:130:1, and the ratio of MSP1T2/POPC in the controlparticle preparation is 1:65.

Synthetic 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) isobtained from Avanti Polar Lipids (Alabaster, Ala.) and prepared as a 75mM (a concentration within the range of 70 to 80 mM is acceptable) stockin chloroform and stored at −20° C. The lipid concentration isdetermined by quantifying total phosphorus using the method of Chen etal. (1956). Amphotericin B (AmB) powder is obtained from Sigma (St.Louis, Mo.) and prepared as 2 mM stock in DMSO, protected from light andstored at −20° C. MSP1T2 is expressed and purified as described byDenisov et al. (2004).

AmB is added to POPC lipid solution to give a lipid:AmB molar ratio of65:1. The solution is dried under a stream of nitrogen and placed undervacuum overnight to remove residual solvent. Manipulations of samplescontaining AmB were protected from light whenever possible. The mixtureis resuspended by the addition of 100 mM cholate in standard buffer (10mM Tris-HCl (pH 7.4), 0.1M NaCl, 1 mM EDTA) to yield a lipidconcentration of 50 mM. The tubes are vortexed, sonicated, and heatedbriefly in a 37° C. water bath, until the mixture is completelysolubilized.

MSP1T2 protein is added to the lipid/AmB/cholate solution to give aprotein/lipid/AmB ratio of 2:130:1. The final lipid concentration isapproximately 15 mM. The mixture is incubated for 2 h at 4° C., near thephase transition temperature for POPC. BioBeads are added to removecholate, and the sample is incubated for an additional 2 h at 4° C.Samples are separated by size exclusion chromatography on a Superdex 200HR 10/30 column (Amersham Biosciences, Piscataway, N.J.), and the 10 nmfractions were retained. Concentration of Amphotericin B is determinedby comparing the A₄₀₅ to a standard curve of Amphotericin B constructedfrom 1 to 20 μg/ml. Nanodisc concentration is determined by measuringA₂₈₀ (MSP1T2 ε₂₈₀=21,000 M⁻¹ cm⁻¹).

Example 15 Ketoconazole-loaded Nanodiscs

Nanodiscs containing the small molecule antifungal ketoconazole wereprepared by incubating MSP1T2, ketoconazole, DMPC, and cholate at amolar ratio of 1:10:80:160, respectively. The mixture was incubated at25° C. for 45 min, BioBeads were added (50% w/v), and incubation wascontinued for an additional 45 min. Incubation with BioBeads removedcholate, and resulted in the self assembly of Nanodiscs and thepartitioning of lipophilic ketoconazole to the bilayer environment ofnascent Nanodiscs. The ketoconazole containing Nanodiscs were purifiedby nickel affinity chromatography and the column eluate was concentratedby diafiltration with Standard Buffer (20 mM Tris-HCl (pH 7.4), 0.1 MNaCl, 0.5 mM EDTA).

Antifungal activity of the ketoconazole-containing Nanodisc preparationwas qualitatively assayed against a lawn of Candida albicans grown onYeast Potato Dextrose (YPD) agar. A colony of freshly grown C. albicanswas mixed in sterile water, and 1 ml of the cell suspension was evenlyapplied to the surface of a YPD-agar plate. The excess cell liquid wasdecanted and 20 μl aliquots of Nanodiscs containing ketoconazole,Nanodiscs prepared without ketoconazole (empty Nanodiscs), and 13 μg/mlketoconazole solution in 1% DMSO were spotted separately onto thesurface of the plate (FIG. 21A). The plate was incubated for 18 hr at35° C. Nanodiscs containing ketoconazole inhibited the growth of the C.albicans in a manner consistent with the ketoconazole control, whereasempty Nanodiscs showed no effect of fungal growth. This resultdemonstrates that the ketoconazole antifungal activity co-purified withthe Nanodiscs and indicates that ketoconazole was associated with theNanodiscs following self-assembly. Application of 20 μl aliquots ofStandard Buffer or 1% DMSO showed no antifungal activity (FIG. 21B).Addition of 20 μl of a 0.13 μg/ml solution of ketoconazole did notvisibly inhibit growth, indicating that the application of this 100-foldlower concentration of the drug was too dilute to affect the fungi atthe cell density present on the plate.

Example 16 Gadolinium-containing Nanodiscs

Nanodiscs containing an amphiphilic gadolinium chelate can be made byincorporating phospholipid or other type of lipid having a chelatinggroup as the polar headgroup portion of the amphiphilic lipid. One suchphospholipid is synthesized by reacting phosphatidylethanolamine withthe dianhydride of diethylenetriaminepentaacetic acid (DTPA) to yield atetradentate chelating phospholipid which can be loaded with Gd³⁺ eitherbefore or after assembly of nanodiscs. The chelating lipid with orwithout bound Gd³⁺ can be mixed with or without other types ofphospholipid in organic solution followed by removal of solvent andformation of nanodiscs by the usual methods.

Another chelating agent suitable for gadolinium cations is1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetate(DPPE-DTPA). An ethylenediaminetetraacetate (EDTA) derivative can beused in place of the DTPA group. Dimyristoyl and distearoyl cansubstitute for the dipalmitoyl moieties. The relatively long chain fattyacids attached to the remainder of the chelating molecule facilitateuptake of the gadolinium or other ion of interest (including but notlimited to trivalent cations of iridium, technecium, and lanthanides ingeneral) into the Nanodisc particles. DTPA phospholipids are useful. SeeUrizzi et al. (1996) Tetrahedron Lett. 37:4685-4688 for a discussion oflipophilic chelating agents.

Example 17 Nanodiscs Containing Photodynamic Compounds

Nanodiscs containing a photodynamic compound, especially a therapeuticphotodynamic compound are prepared essentially as described herein abovefor Amphotericin B and ketoconazole but with the use of the photodynamiccompound, such as a psoralen, phthalacyanin or porphyrin, with themodification that stock solutions, assembly reactions and Nanodiscpreparations are protected from light.

Example 18 Fluorescently Labeled Nanodiscs

The methodology for preparation of Nanodiscs containing small organicmolecules, in particular fluorescent labels is as follows.

All glassware involved in this procedure is to be washed with 1M KOH andsonicated for 15 minutes when possible.

Two Nanodisc preparations, one labeled and one unlabeled are described.The ratio of MSP1/DPPC/Dil in the labeled prep is 1/100/.05 with 0.5 mgof MSP1 used, and the ratio of MSP1/DPPC in the unlabeled prep is 1/100with 2 mg of MSP1 used. DPPC is obtained from stock solutions dissolvedin chloroform. Appropriate amounts of DPPC are delivered to two glasstubes. Dil in ethanol is added to the labeled disc prep tube. Solvent isdried down using nitrogen, and samples are placed in a vacuum dessicatorovernight to remove residual solvent.

50 mM cholate in standard buffer (10 mM Tris-HCl pH 7.4, 0.1M NaCl, 1 mMEDTA, 0.01% NaN₃) is added to the dried lipid samples to yield 25 mMfinal lipid concentration. The tubes are vortexed, heated, and sonicateduntil lipid is completely in solution. 0.5 mg of MSP1 in buffer at 37°C. is added to the sample to be labeled, and 2 mg of MSP1 in buffer at37° C. is added to the unlabeled sample. The samples are incubated at37° C. for 4 hours. Dialysis is then performed to remove the sodiumcholate. Dialysis is done at 37° C. in standard buffer for 24 hours with3 buffer changes.

Both samples are concentrated to about 0.3 ml, filtered, and subjectedto size exclusion chromatography on a Superdex column. Fractions arecollected, combining and saving those which contain particles with adiameter of about 10 nm. Concentration of the Nanodiscs is determined bymeasuring absorption at 280 nm (A₂₈₀ of 1 mg/ml MSP1 is 1.0). Absorptionof Dil at 280 nm is assumed to be negligible in the labeled disc sample.

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1. An immunogenic composition comprising stable, soluble nanoscalediscoid particles, which particles comprise a membrane scaffold protein,at least one hydrophobic or partially hydrophobic antigen molecule froma virus, a bacterium, fungus, protozoan, parasite, a human neoplasticcell or an animal neoplastic, tumor or cancer cell, and at least onephospholipid, wherein said immunogenic composition optionally furthercomprises an immunological adjuvant, and wherein said membrane scaffoldprotein is a derivative or a truncated form of human apolipoprotein A1,is amphipathic and wherein said membrane scaffold protein forms at leastone alpha helix, and lacks the N-terminal globular domain of humanapolipoprotein A1 (amino acids 8-50 of SEQ ID NO:2) and wherein themembrane scaffold protein is that of SEQ ID NO:6 or SEQ ID NO:9.
 2. Theimmunogenic composition of claim 1, wherein the hydrophobic or partiallyhydrophobic antigen molecule is from a solubilized membrane preparationor a solubilized membrane fragment preparation.
 3. The immunogeniccomposition of claim 1, wherein said hydrophobic or partiallyhydrophobic antigen molecule is a protein, a lipoprotein,lipopolysaccharide, lipooligosaccharide, glycoprotein, or a glycolipid.4. The immunogenic composition of claim 1, wherein said artificialmembrane scaffold protein comprises the amino acid sequence as set forthin SEQ ID NO:
 6. 5. The immunogenic composition of claim 1, wherein thebacterium is a rickettsia or a mycoplasma.
 6. The immunogeniccomposition of claim 1, wherein the antigenic molecule is selected fromthe group consisting of gp120 of Human Immunodeficiency Virus, anenvelope glycoprotein of Herpes simplex virus, an envelope glycoproteinof measles virus, a “spike” protein of SARS virus, a hemagglutinin ofinfluenza virus, a hemagglutinin of parainfluenza virus, an M6 proteinof Streptococcus pyogenes, a fimbrillin of Porphoryomonas gingivalis, anInIB protein of Listeria monocytogenes, an ActA protein of Listeriamonocytogenes, a YadA protein of Yersinia enterocolitica, an IcsAprotein of Shigella flexneri, an invasin of Yersinia pseudotuberculosis,at least one acf gene product of Vibrio cholerae, capsular materialcomprising the poly-D-glutamate polypeptide of Bacillus anthracis, afibrinogen/fibrin binding protein of Staphylococcus aureus, V and/or Wantigens of Yersinia pestis, Yersinia enterocolitica or Yersiniapseudotuberculosis, flagellin of Campylobacter jejuni, a porin ofCampylobacter jejuni, and an O antigen of Salmonella typhi, Salmonellacholeraesuis or Salmonella enteritidis.
 7. The immunogenic compositionof claim 1, wherein the human neoplastic cell or animal neoplastic cellis a malignant melanoma cell, a gastrointestinal carcinoma cell, aneuroblastoma cell, an osteosarcoma cell, a renal carcinoma cell, abreast carcinoma cell, a lung carcinoma cell, a leukemia cell, alymphoma cell or a myeloma cell.
 8. The immunogenic composition of claim1, wherein the nanoscale particle further comprises a molecule whichtargets said nanoscale particle to a cell surface.
 9. The immunogeniccomposition of claim 8, wherein the cell surface is a mucosal cellsurface.
 10. The immunogenic composition of claim 9, wherein saidmucosal cell surface is an intestinal mucosal cell surface.
 11. Theimmunogenic composition of claim 8, wherein the cell surface is anepithelial cell surface.
 12. The immunogenic composition of claim 1,wherein the at least one phospholipid is phosphatidyl choline,phosphatidyl ethanolamine, phosphatidyl inositol,dipalmitoyl-phosphatidylcholine, dimyristoyl phosphatidyl choline,1-palmitoyl-2-oleoyl-phosphatidyl choline,1-palmitoyl-2-oleoyl-phosphatidyl serine,1-palmitoyl-2-oleoyl-phosphatidyl ethanolamine, dihexanoyl phosphatidylcholine, dipalmitoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidylinositol, dimyristoyl phosphatidyl ethanolamine, dimyristoylphosphatidyl inositol, dihexanoyl phosphatidyl ethanolamine, dihexanoylphosphatidyl inositol, 1-palmitoyl-2-oleoyl-phosphatidyl ethanolamineand 1-palmitoyl-2-oleoyl-phosphatidyl inositol.
 13. The immunogeniccomposition of claim 1, wherein said artificial membrane scaffoldprotein comprises the amino acid sequence as set forth in SEQ ID NO: 9.